Nucleic Acid Sequences Encoding Proteins Associated with Abiotic Stress Responses and Plant Cells with Increased Tolerance to Environmental Stress

ABSTRACT

This invention relates generally to nucleic acid sequences encoding proteins that are associated with abiotic stress responses and abiotic stress tolerance in plants. This invention further relates to transformed plant cells with altered metabolic activity compared to a corresponding non transformed wild type plant cell, wherein the metabolic activity is altered by transformation with a Stress-Related Protein (SRP) coding nucleic acid and results in increased tolerance and/or resistance to an environmental stress as compared to a corresponding non-transformed wild type plant cell.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/907,438, filed Oct. 19, 2010, which is a divisional of U.S.application Ser. No. 11/251,208, filed Oct. 14, 2005, now U.S. Pat. No.7,847,159, which is a continuation-in-part of PCT/US2004/011888 filedApr. 15, 2004, which claims benefit to European application 03008080.8,filed Apr. 15, 2003, European application 03009728.1, filed May 2, 2003,European application 03016672.2, filed Aug. 1, 2003, and Europeanapplication 03022225.1, filed Sep. 30, 2005. The entire content of eachof the aforementioned applications is hereby incorporated by referencein its entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_(—)13311_(—)00086. The size ofthe text file is 1,890 KB, and the text file was created on Jul. 16,2012.

This invention relates generally to nucleic acid sequences encodingproteins that are associated with abiotic stress responses and abioticstress tolerance in plants.

This invention further relates to transformed plant cells with alteredmetabolic activity compared to a corresponding non transformed wild typeplant cell, wherein the metabolic activity is altered by transformationwith a Stress-Related Protein (SRP) coding nucleic acid and results inincreased tolerance and/or resistance to an environmental stress ascompared to a corresponding non-transformed wild type plant cell.

In particular, this invention relates to nucleic acid sequences encodingproteins that confer drought, heat, cold, and/or salt tolerance and/orresistance to plants, especially by altering the metabolic activityleading to drought, heat, cold, and/or salt tolerance and/or resistanceto plants. The invention also deals with methods of producing, screeningfor and breeding such plant cells or plants and method of detectingstress in plants cells or plants.

Abiotic environmental stresses such as drought stress, salinity stress,heat stress and cold stress, are major limiting factors of plant growthand productivity (Boyer. 1982. Science 218, 443-448). Crop losses andcrop yield losses of major crops such as rice, maize (corn) and wheatcaused by these stresses represent a significant economic and politicalfactor and contribute to food shortages in many underdevelopedcountries.

Plants are typically exposed during their life cycle to conditions ofreduced environmental water content. Most plants have evolved strategiesto protect themselves against these conditions of low water ordesiccation (drought). However, if the severity and duration of thedrought conditions are too great, the effects on plant development,growth and yield of most crop plants are profound. Continuous exposureto drought causes major alterations in the plant metabolism. These greatchanges in metabolism ultimately lead to cell death and consequentlyyield losses.

Developing stress-tolerant plants is a strategy that has the potentialto solve or mediate at least some of these problems (McKersie andLeshem, 1994. Stress and Stress Coping in Cultivated Plants, KluwerAcademic Publishers). However, traditional plant breeding strategies todevelop new lines of plants that exhibit resistance (tolerance) to thesetypes of stresses are relatively slow and require specific resistantlines for crossing with the desired line. Limited germplasm resourcesfor stress tolerance and incompatibility in crosses between distantlyrelated plant species represent significant problems encountered inconventional breeding. Additionally, the cellular processes leading todrought, cold and salt tolerance are complex in nature and involvemultiple mechanisms of cellular adaptation and numerous metabolicpathways (McKersie and Leshem, 1994. Stress and Stress Coping inCultivated Plants, Kluwer Academic Publishers). This multi-componentnature of stress tolerance has not only made breeding for tolerancelargely unsuccessful, but has also limited the ability to geneticallyengineer stress tolerance plants using biotechnological methods.

Drought, heat, cold and salt stresses have a common theme important forplant growth and that is water availability. Plants are exposed duringtheir entire life cycle to conditions of reduced environmental watercontent. Most plants have evolved strategies to protect themselvesagainst these conditions. However, if the severity and duration of thedrought conditions are too great, the effects on plant development,growth and yield of most crop plants are profound. Since high saltcontent in some soils result in less available water for cell intake,its effect is similar to those observed under drought conditions.Additionally, under freezing temperatures, plant cells loose water as aresult of ice formation that starts in the apoplast and withdraws waterfrom the symplast (McKersie and Leshem, 1994. Stress and Stress Copingin Cultivated Plants, Kluwer Academic Publishers). Commonly, a plant'smolecular response mechanisms to each of these stress conditions aresimilar.

The results of current research indicate that drought tolerance is acomplex quantitative trait and that no real diagnostic marker isavailable yet. High salt concentrations or dehydration may cause damageat the cellular level during drought stress but the precise injury isnot entirely clear (Bray, 1997. Trends Plant Sci. 2, 48-54). This lackof a mechanistic understanding makes it difficult to design a transgenicapproach to improve drought tolerance. However, an important consequenceof damage may be the production of reactive oxygen radicals that causecellular injury, such as lipid peroxidation or protein and nucleic acidmodification. Details of oxygen free radical chemistry and theirreaction with cellular components such as cell membranes have beendescribed (McKersie and Leshem, 1994. Stress and Stress Coping inCultivated Plants, Kluwer Academic Publishers).

It is the object of this invention to identify new, unique genes capableof conferring stress tolerance to plants upon expression orover-expression.

It is further object of this invention to identify, produce and breednew, unique stress tolerant and/or resistant plant cells or plants andmethods of inducing and detecting stress tolerance and/or resistance inplants or plant cells. It is a further object to identify new methods todetect stress tolerance and/or resistance in plants or plant cells. Itis also the object of this invention to identify new, unique genescapable of conferring stress tolerance to plants upon expression orover-expression.

DETAILED DESCRIPTION OF THE DRAWING

The drawing shows the protein alignment of Rho small GTPases from Oryzasativa cv. Noppon-Brarre (a japonica rice), Brassica napus cv. “ACExcel” “Quantum” and “Cresor” (canola), and Glycine max cv. Resuick(soybean).

The present invention provides a transformed plant cell with alteredmetabolic activity compared to a corresponding non transformed wild typeplant cell, wherein the metabolic activity is altered by transformationwith a Stress-Related Protein (SRP) coding nucleic acid and results inincreased tolerance and/or resistance to an environmental stress ascompared to a corresponding non-transformed wild type plant cell.

The present invention provides a transgenic plant cell transformed byStress-Related Protein (SRP) coding nucleic acid. selected from thegroup consisting of:

-   -   a) nucleic acid molecule encoding one of the polypeptides shown        in FIGS. 1 a, 1 b or 1 c or a fragment thereof, which confers an        an altered metabolic activity in an organism or a part thereof;    -   b) nucleic acid molecule comprising one of the nucleic acid        molecule shown in FIGS. 1 a, 1 b or 1 c;    -   c) nucleic acid molecule whose sequence can be deduced from a        polypeptide sequence encoded by a nucleic acid molecule of (a)        or (b) as a result of the degeneracy of the genetic code and        conferring an altered metabolic activity in an organism or a        part thereof;    -   d) nucleic acid molecule which encodes a polypeptide which has        at least 50% identity with the amino acid sequence of the        polypeptide encoded by the nucleic acid molecule of (a) to (c)        and conferring an altered metabolic activity in an organism or a        part thereof;    -   e) nucleic acid molecule which hybridizes with a nucleic acid        molecule of (a) to (c) under stringent hybridisation conditions        and conferring an altered metabolic activity in an organism or a        part thereof;    -   f) nucleic acid molecule which encompasses a nucleic acid        molecule which is obtained by amplifying nucleic acid molecules        from a cDNA library or a genomic library using the primers as        shown in table 2 and conferring an altered metabolic activity in        an organism or a part thereof;    -   g) nucleic acid molecule encoding a polypeptide which is        isolated with the aid of monoclonal antibodies against a        polypeptide encoded by one of the nucleic acid molecules of (a)        to (f) and conferring an altered metabolic activity in an        organism or a part thereof;    -   h) nucleic acid molecule encoding a polypeptide comprising the        consensus sequence shown in FIG. 2 and conferring an altered        metabolic activity in an organism or a part thereof; and    -   i) nucleic acid molecule which is obtainable by screening a        suitable nucleic acid library under stringent hybridization        conditions with a probe comprising one of the sequences of the        nucleic acid molecule of (a) to (h) or with a fragment thereof        having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,        200 nt or 500 nt of the nucleic acid molecule characterized        in (a) to (h) and conferring an altered metabolic activity in an        organism or a part thereof.    -   or comprising a sequence which is complementary thereto.

For the purpose of the present invention the term “FIGS. 1 a, 1 b or 1c” involves and means the SEQ ID NO: 1 to 556 and the term “consensussequence shown in FIG. 2” means SEQ ID NO: 557 to 560.

Particularly, the term “FIG. 1 a” means SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269 and/or 270,the term “FIG. 1 b” means SEQ ID NO: 271, 272, 273, 274, 275, 276, 277,278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 and/or 290,the term “FIG. 1 c” means SEQ ID NO: 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381,382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409,410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437,438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479,480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493,494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507,508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521,522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535,536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549,550, 551, 552, 553, 554, 555 and/or 556,and the term “consensus sequence shown in FIG. 2” means SEQ ID NO: 557,558, 559 and/or 560.

More precisely, when “polypeptides or proteins according to FIG. 1 a”are mentioned, then the SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206,208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262,264, 266, 268 and/or 270 are meant;

when “polypeptides or proteins according to FIG. 1 b” are mentioned,then the SEQ ID NO 272, 274, 276, 278, 280, 282, 284, 286, 288 and/or290 are meant;when “polypeptides or proteins according to FIG. 1 c” are mentioned,then the SEQ ID NO 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339,341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367,369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395,397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423,425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451,453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479,481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507,509, 511, 513, 515, 517, 519, 521, 523, 526, 528, 530, 532, 534, 536,538, 540, 542, 544, 546, 548, 550, 552, 554 and/or 556 are meant. Moreprecisely, when “polynucleotides or nucleic acid molecules according toFIG. 1 a” are mentioned, then the SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257,259, 261, 263, 265, 267 and/or 269 are meant;when “polynucleotides or nucleic acid molecules according to FIG. 1 b”are mentioned, then the SEQ ID NO 271, 273, 275, 277, 279, 281, 283,285, 287 and/or 289 are meant;when “polynucleotides or nucleic acid molecules according to FIG. 1 c”are mentioned, then the SEQ ID NO 291, 293, 295, 297, 299, 301, 303,305, 307, 309, 311, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330,332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386,388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414,416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442,444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470,472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498,500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 525,527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553and/or 555 are meant.

As used herein, the term “metabolite” refers to intermediate substances,preferably such of low molecular weight, which occur during anabolismand catabolism in a cell or plant, in other words a substance producedor concumed by metabolism.

The term “altered metabolic activity” refers to the change (increase ordecrease) of the amount, concentration or activity (meaning here theeffective concentration for the purposes of chemical reactions and othermass action) of a metabolite in a specific volume relative to acorresponding volume (e.g. in an organism, a tissue, a cell or a cellcompartment) of a control, reference or wild type, including the de novocreation of the activity or expression, measured for example by one ofthe methods described herein below, which is changed (increased ordecreased) as compared to a corresponding non transformed wild typeplant cell.

The terms “increased”, “rised”, “extended”, “enhanced”, “improved” or“amplified” relate to a corresponding change of a property in anorganism, a part of an organism such as a tissue, seed, root, leave,flower etc. or in a cell and are interchangeable. Preferrably, theoverall activity in the volume is increased or enhanced in cases if theincrease or enhancement is related to the increase or enhancement of anactivity of a gene product, independent whether the amount of geneproduct or the specific activity of the gene product or both isincreased or enhanced or whether the amount, stability or translationefficacy of the nucleic acid sequence or gene encoding for the geneproduct is increased or enhanced. The terms “reduction”, “decrease” or“deletion” relate to a corresponding change of a property in anorganism, a part of an organism such as a tissue, seed, root, leave,flower etc. or in a cell. Preferrably, the overall activity in thevolume is reduced, decreased or deleted in cases if the reduction,decrease or deletion is related to the reduction, decrease or deletionof an activity of a gene product, independent whether the amount of geneproduct or the specific activity of the gene product or both is reduced,decreased or deleted or whether the amount, stability or translationefficacy of the nucleic acid sequence or gene encoding for the geneproduct is reduced, decreased or deleted.

The terms “increase” or “decrease” relate to a corresponding change of aproperty in an organism or in a part of an organism, such as a tissue,seed, root, leave, flower etc. or in a cell. Preferrably, the overallactivity in the volume is increased in cases the increase relates to theincrease of an activity of a gene product, independent whether theamount of gene product or the specific activity of the gene product orboth is increased or generated or whether the amount, stability ortranslation efficacy of the nucleic acid sequence or gene encoding forthe gene product is increased.

Under “change of a property” it is understood that the activity,expression level or amount of a gene product or the metabolite contentis changed in a specific volume relative to a corresponding volume of acontrol, reference or wild type, including the de novo creation of theactivity or expression.

The terms “increase” or “decrease” include the change of said propertyin only parts of the subject of the present invention, for example, themodification can be found in compartment of a cell, like a organelle, orin a part of a plant, like tissue, seed, root, leave, flower etc. but isnot detectable if the overall subject, i.e. complete cell or plant, istested. Preferably, the increase or decrease is found cellular, thus theterm “increase of an activity” or “increase of a metabolite content”relates to the cellular increase compared to the wild type cell.

Accordingly, the term “increase” or “decrease” means that the specificactivity of an enzyme as well as the amount of a compound or metabolite,e.g. of a polypeptide, a nucleic acid molelcule or of the fine chemicalof the invention or an encoding mRNA or DNA, can be increased ordecreased in a volume.

The terms “wild type”, “control” or “reference” are exchangeable and canbe a cell or a part of organisms such as an organelle or a tissue, or anorganism, in particular a microorganism or a plant, which was notmodified or treated according to the herein described process accordingto the invention. Accordingly, the cell or a part of organisms such asan organelle or a tissue, or an organism, in particular a microorganismor a plant used as wild type, control or reference corresponds to thecell, organism or part thereof as much as possible and is in any otherproperty but in the result of the process of the invention as identicalto the subject matter of the invention as possible. Thus, the wild type,control or reference is treated identically or as identical as possible,saying that only conditions or properties might be different which donot influence the quality of the tested property.

Preferably, any comparison is carried out under analogous conditions.The term “analogous conditions” means that all conditions such as, forexample, culture or growing conditions, assay conditions (such as buffercomposition, temperature, substrates, pathogen strain, concentrationsand the like) are kept identical between the experiments to be compared.

The “reference”, “control”, or “wild type” is preferably a subject, e.g.an organelle, a cell, a tissue, an organism, in particular a plant or amicroorganism, which was not modified or treated according to the hereindescribed process of the invention and is in any other property assimilar to the subject matter of the invention as possible.

The reference, control or wild type is in its genome, transcriptome,proteome or meta-bolome as similar as possible to the subject of thepresent invention. Preferably, the term “reference-” “control-” or “wildtype-”-organelle, -cell, -tissue or -organism, in particular plant ormicroorganism, relates to an organelle, cell, tissue or organism, inparticular plant or microorganism, which is nearly genetically identicalto the organelle, cell, tissue or organism, in particular microorganismor plant, of the present invention or a part thereof preferably 95%,more preferred are 98%, even more preferred are 99.00%, in particular99.10%, 99.30%, 99.50%, 99.70%, 99.90%, 99.99%, 99.999% or more. Mostpreferable the “reference”, “control”, or “wild type” is a subject, e.g.an organelle, a cell, a tissue, an organism, which is geneticallyidentical to the organism, cell or organelle used according to theprocess of the invention except that the responsible or activityconferring nucleic acid molecules or the gene product encoded by themare amended, manipulated, exchanged or introduced according to theinventive process.

Preferably, the reference, control or wild type differs form the subjectof the present invention only in the cellular activity of thepolypeptide of the invention, e.g. as result of an increase in the levelof the nucleic acid molecule of the present invention or an increase ofthe specific activity of the polypeptide of the invention, e.g. by or inthe expression level or activity of an protein having the activity of anStress-Related Protein (SRP) or its homologs, its biochemical orgenetical causes and the altered metabolic activity.

In case, a control, reference or wild type differing from the subject ofthe present invention only by not being subject of the process of theinvention can not be provided, a control, reference or wild type can bean organism in which the cause for the modulation of an activityconferring the altered metabolic activity or expression of the nucleicacid molecule of the invention as described herein has been switchedback or off, e.g. by knocking out the expression of responsible geneproduct, e.g. by antisense inhibition, by inactivation of an activatoror agonist, by activation of an inhibitor or antagonist, by inhibitionthrough adding inhibitory antibodies, by adding active compounds as e.g.hormones, by introducing negative dominant mutants, etc. A geneproduction can for example be knocked out by introducing inactivatingpoint mutations, which lead to an enzymatic activity inhibition or adestabilization or an inhibition of the ability to bind to cofactorsetc.

Accordingly, preferred reference subject is the starting subject of thepresent process of the invention. Preferably, the reference and thesubject matter of the invention are compared after standardization andnormalization, e.g. to the amount of total RNA, DNA, or protein oractivity or expression of reference genes, like housekeeping genes, suchas ubiquitin, actin or ribosomal proteins.

A series of mechanisms exists via which a modification of the a protein,e.g. the polypeptide of the invention can directly or indirectly affectthe yield, production and/or production efficiency of the amino acid.

For example, the molecule number or the specific activity of thepolypeptide or the nucleic acid molecule may be increased. Largeramounts of the fine chemical can be produced if the polypeptide or thenucleic acid of the invention is expressed de novo in an organismlacking the activity of said protein. However, it is also possible toincrease the expression of the gene which is naturally present in theorganisms, for example by modifying the regulation of the gene, or byincreasing the stability of the corresponding mRNA or of thecorresponding gene product encoded by the nucleic acid molecule of theinvention, or by introducing homologous genes from other organisms whichare differently regulated, eg. not feedback sensitive.

This also applies analogously to the combined increased expression ofthe nucleic acid molecule of the present invention or its gene productwith that of further enzymes of the amino acid biosynthesis pathways,e.g. which are useful for the synthesis of the fine chemicals.

The increase, decrease or modulation according to this invention can beconstitutive, e.g. due to a stable permanent transgenic expression or toa stable mutation in the corresponding endogenous gene encoding thenucleic acid molecule of the invention or to a modulation of theexpression or of the behaviour of a gene conferring the expression ofthe polypeptide of the invention, or transient, e.g. due to an transienttransformation or temporary addition of a modulator such as a agonist orantagonist or inducible, e.g. after transformation with a inducibleconstruct carrying the nucleic acid molecule of the invention undercontrol of a induceable promoter and adding the inducer, e.g.tetracycline or as described herein below.

The increase in activity of the polypeptide amounts in a cell, a tissue,a organelle, an organ or an organism or a part thereof preferably to atleast 5%, preferably to at least 20% or at to least 50%, especiallypreferably to at least 70%, 80%, 90% or more, very especially preferablyare to at least 200%, most preferably are to at least 500% or more incomparison to the control, reference or wild type.

The specific activity of a polypeptide encoded by a nucleic acidmolecule of the present invention or of the polypeptide of the presentinvention can be tested as described in the examples. In particular, theexpression of a protein in question in a cell, e.g. a plant cell or amicroorganism and the detection of an increase the fine chemical levelin comparison to a control is an easy test and can be performed asdescribed in the state of the art.

The term “increase” includes, that a compound or an activity isintroduced into a cell de novo or that the compound or the activity hasnot been detectable before, in other words it is “generated”.

Accordingly, in the following, the term “increasing” also comprises theterm “generating” or “stimulating”. The increased activity manifestsitself in an increase of the fine chemical.

The transformed plant cells are compared to the correspondingnon-transformed wild type of the same genus and species under otherwiseidentical conditions (such as, for example, culture conditions, age ofthe plants and the like). In this context, a change in metabolicactivity of at least 10%, advantageously of at least 20%, preferably atleast 30%, especially preferably of at least 40%, 50% or 60%, veryespecially preferably of at least 70%, 80%, 90%, 95% or even 100% ormore, in comparison with the non-transformed organism is advantageous.

Preferably the change in metabolite concentration of the transformedplant cells is the changed compared to the corresponding non-transformedwild type. Preferably the change in metabolite concentration is measuredby HPLC and calculated by dividing the peak height or peak area of eachanalyte (metabolite) through the peak area of the respective internalstandards. Data is normalised using the individual sample fresh weight.The resulting values are divided by the mean values found for wild typeplants grown under control conditions and analysed in the same sequence,resulting in the so-called ratios, which represent values independent ofthe analytical sequence. These ratios indicate the behavior of themetabolite concentration of the transformed plants in comparison to theconcentration in the wild type control plants.

According to this method, the change in at least one metaboliteconcentration of the transformed plant cells compared to thecorresponding non-transformed wild type is at least 10%, advantageouslyof at least 20%, preferably at least 40%, 60% or 80%, especiallypreferably of at least 90%, 100% or 200%, very especially preferably ofat least 700%, 800%, 900% 1000% or more.

Data significance can be determinated by all statistical methods knownby a person skilled in the art, preferably by a t-test, more preferablyby the student t-test.

Altered metabolic activity also refers to metabolites that, compared toa corresponding non transformed wild type plant cell, are not producedafter transformation or are only produced after transformation.

Preferred metabolites of the invention are 2,3-dimethyl-5-phytylquinolor 2-hydroxy-palmitic acid or 3,4-dihydroxyphenylalanine (=dopa) or3-hydroxy-palmitic acid or 5-oxoproline or alanine or alpha linolenicacid (c18:3 (c9, c12, c15)) or alpha-tocopherol or aminoadipic acid oranhydroglucose or arginine or aspartic acid or beta-apo-8′ carotenal orbeta-carotene or beta-sitosterol or beta-tocopherol or(delta-7-cis,10-cis)-hexadecadienic acid or hexadecatrienic acid ormargaric acid or delta-15-cis-tetracosenic acid or ferulic acid orcampesterol or cerotic acid (c26:0) or citrulline or cryptoxanthine oreicosenoic acid (20:1) or fructose or fumarate or galactose orgamma-aminobutyric acid or gamma-tocopherol or gluconic acid or glucoseor glutamic acid or glutamine or glycerate or glycerinaldehyd orglycerol or glycerol-3-phosphate or glycine or homoserine or inositol orisoleucine or iso-maltose or isopentenyl pyrophosphate or leucine orlignoceric acid (c24:0) or linoleic acid (c18:2 (c9, c12)) or luteine orlycopene or malate or mannose or methionine or methylgalactofuranosideor methylgalactopyranoside or methylgalactopyranoside or palmitic acid(c16:0) or phenylalanine or phosphate or proline or putrescine orpyruvat or raffinose or ribonic acid or serine or shikimate or sinapineacid or stearic acid (c18:0) or succinate or sucrose or threonine ortriacontanoic acid or tryptophane or tyrosine or ubichinone orudp-glucose or valine or zeaxanthine.

Metabolic activity may also be altered concerning one or more derivatesof one or more of the above metabolites.

Preferably metabolic activity is altered concerning one or moremetabolites selected from the group consisting of all of the abovemetabolites.

Alternatively metabolic activity may be altered concerning one or moremetabolites selected from the group consisting of mannose, inositol,phosphate, aspartic acid, isoleucine, leucine, gamma-aminobutyric acid,glycerinaldehyd, sucrose, campesterol, valine, beta-tocopherol,oubichinone, palmitic acid (c16:0), 2-hydroxy-palmitic acid,2,3-dimethyl-5-phytylquinol, beta-carotene, alpha-linolenic acid (c18:3(c9, c12, c15)), lycopene.

Alternatively metabolic activity may be altered concerning one or moremetabolites selected from the group consisting ofmethylgalactofuranoside, beta-sitosterol, delta-15-cis-tetracosenic acid(c24:1 me), margaric acid (c17:0 me), stearic acid (c18:0),methylgalactopyranoside, gamma-tocopherol, linoleic acid (c18:2 (c9,c12)), hexadecatrienic acid (c16:3 me), shikimate, raffinose, glutamicacid, glutamine, udp-glucose, proline, threonine, isopentenylpyrophosphate, 5-oxoproline, ferulic acid, sinapine acid.

Alternatively metabolic activity may be altered concerning one or moremetabolites selected from the group consisting of tryptophane,citrulline, serine, alanine, glycerate, arginine, 3-hydroxy-palmiticacid, putrescine, 3,4-dihydroxyphenylalanine (=dopa), alpha-tocopherol,aminoadipic acid, anhydroglucose, beta-apo-8′ carotenal,delta-7-cis,10-cis-hexadecadienic acid (c16:2 me), cerotic acid (c26:0),cryptoxanthine, eicosenoic acid (20:1), fructose, fumarate.

Alternatively metabolic activity may be altered concerning one or moremetabolites selected from the group consisting of galactose, gluconicacid, glucose, glycerol, glycerol-3-phosphate, glycine, homoserine,iso-maltose, lignoceric acid (c24:0), luteine, malate, triacontanoicacid, methionine, phenylalanine, pyruvate, ribonic acid, succinate,tyrosine, zeaxanthine.

The present invention provides a transgenic plant cell, whereinexpression of said nucleic acid sequence in the plant cell resultsaltered metabolic activity leading to increased tolerance and/orresistance to environmental stress as compared to a correspondingnon-transformed wild type plant cell. One preferred wild type plant cellis a non-transformed Arabidopsis plant cell. An example here is theArabidopsis wild type C24 (Nottingham Arabidopsis Stock Centre, UK; NASCStock N906).

Other preferred wild type plant cells are a non-transformed from plantsselected from the group consisting of maize, wheat, rye, oat, triticale,rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot,pepper, sunflower, flax, borage, safflower, linseed, primrose, rapeseed,turnip rape, tagetes, solanaceous plants, potato, tobacco, eggplant,tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species,oil palm, coconut, perennial grass and forage crops.

More preferred wild type plant cells are a non-transformed Linum plantcell, preferably Linum usitatissimum, more preferably the varietyBrigitta, Golda, Gold Merchant, Helle, Juliel, Olpina, Livia, Marlin,Maedgold, Sporpion, Serenade, Linus, Taunus, Lifax or Liviola, anon-transformed Heliantus plant cell, preferably Heliantus annuus, morepreferably the variety Aurasol, Capella, Flavia, Flores, Jazzy, Palulo,Pegasol, PIR64° A.54, Rigasol, Sariuca, Sideral, Sunny, Alenka, Candisolor Floyd, or a non-transformed Brassica plant cell, preferably Brassicanapus, more preferably the variety Dorothy, Evita, Heros, Hyola, Kimbar,Lambada, Licolly, Liconira, Licosmos, Lisonne, Mistral, Passat, Serator,Siapula, Sponsor, Star, Caviar, Hybridol, Baical, Olga, Lara, Doublol,Karola, Falcon, Spirit, Olymp, Zeus, Libero, Kyola, Licord, Lion,Lirajet, Lisbeth, Magnum, Maja, Mendel, Mica, Mohican, Olpop, Ontarion,Panthar, Prinoe, Pronio, Susanna, Talani, Titan, Transfer, Wiking,Woltan, Zeniah, Artus, Contact or Smart.

The expression of said nucleic acid sequence in the plant cell maydirectly or indirectly influence the metabolic activity of thetransformed plant cells. Preferably they influence the activity of theabove metabolites.

Preferably metabolic activity may be altered by transformation with oneor more Stress-Related Protein (SRP) coding nucleic acid selected fromthe group comprising the nucleic acid of FIGS. 1 a, 1 b or 1 c homologsof the aforementioned sequences.

It is within the scope of the invention to identify the genes encoded bya nucleic acid sequence selected from the group consisting of thenucleic acid of FIGS. 1 a, 1 b or 1 c and/or homologs thereof in targetplants, especially crop plants, and then express the corresponding geneto achieve the altered metabolic activity resulting in increasedtolerance and/or resistance to environmental stress. Consequently theinvention is not limited to a specific plant.

A protein having an activity conferring an altered metabolic activitypreferably has the structure of the polypeptide described herein, inparticular of the polypeptides comprising the consensus sequence shownin FIG. 2 or of the polypeptide as shown in FIGS. 1 a, 1 b or 1 c or thefunctional homologues thereof as described herein, or is encoded by thenucleic acid molecule characterized herein or the nucleic acid moleculeaccording to the invention, for example by the nucleic acid molecule asshown in FIGS. 1 a, 1 b or 1 c or its herein described functionalhomologues and has the herein mentioned activity.

It is further possible to detect environmental stress in plant cells orplants by screening the plant cells for altered metabolic activity ascompared to non-stress conditions. This allows for monitoring of stresslevels in plants, even when no symptoms are visible. Therefore counteraction can be taken ealier and e.g. crop losses minimized by timelywatering.

It is also within the scope of the invention to screen plant cells orplants for increased tolerance and/or resistance to environmental stressby screening the plant cells under stress conditions for alteredmetabolic activity as compared to non-stress conditions. This allowsselection of plants with increased tolerance and/or resistance toenvironmental stress without the identification of genes or visualsymptoms.

With the invention it is further possible to breed plant cells or plantstowards increased tolerance and/or resistance to environmental stress byscreening the plant cells under stress conditions for altered metabolicactivity as compared to non-stress conditions and selecting those withincreased tolerance and/or resistance to environmental stress. Thescreening for metabolite activity is faster and easier than e.g.screening for genes.

Screening is well known to those skilled in the art and generally refersto the search for a particular attribute or trait. In the invention thistrait in a plant or plant cell is preferably the concentration of ametabolite, especially preferred the concentration of the abovemetabolites. The methods and devices for screening are familiar to thoseskilled in the art and include GC (gas chromatography), LC (liquidchromatography), HPLC (high performance (pressure) liquidchromatography), MS (mass spectrometry), NMR (nuclear magneticresonance) spectroscopy, IR (infra red) spectroscopy, photometricmethods etc and combinations of these methods.

Breeding is also customary knowledge for those skilled in the art. It isunderstood as the directed and stable incorporation of a particularattribute or trait into a plant or plant cell.

The various breeding steps are characterized by well-defined humanintervention such as selecting the lines to be crossed, directingpollination of the parental lines, or selecting appropriate progenyplants. Different breeding measures can be taken, depending on thedesired properties. All the techniques are well known by a personskilled in the art and include for example, but are not limited tohybridization, inbreeding, backcross breeding, multiline breeding,variety blend, interspecific hybridization, aneuploid techniques, etc.Hybridization techniques also can include the sterilization of plants toyield male or female sterile plants by mechanical, chemical, orbiochemical means. Cross pollination of a male sterile plant with pollenof a different line assures that the genome of the male sterile butfemale fertile plant will uniformly obtain properties of both of theparental lines. The transgenic seeds and plants according to theinvention can therefor be used for the breeding of improved plant lines,which can increase the effectiveness of conventional methods such asherbicide or pesticide treatment or which allow one to dispense withsaid methods due to their modified genetic properties. Alternatively newcrops with improved stress tolerance, preferably drought andtemperature, can be obtained, which, due to their optimized genetic“equipment”, yield harvested product of better quality than productsthat were not able to tolerate comparable adverse developmentalconditions.

The invention provides that the environmental stress can be salinity,drought, temperature, metal, chemical, pathogenic and oxidativestresses, or combinations thereof, preferably drought and/ortemperature.

The object of the invention is a transgenic plant cell, wherein the SRP(=stress related protein) is selected preferably from yeast, preferablySaccharomyces cerevisiae, or E. coli or a plant, preferably Brassicanapus, Glycine max, or Oryza sativa.

Object of the invention is also a transgenic plant cell, wherein the SRPcoding nucleic acid is at least about 50% homologous to one of thenucleic acid of FIGS. 1 a, 1 b or 1 c.

In the transgenic plant cell of the invention, the expression of saidnucleic acid results in increased tolerance to an environmental stress,which is preferably achieved by altering metabolic activity, as comparedto a corresponding non-transformed wild type plant cell. Herein, theenvironmental stress is selected from the group consisting of salinity,drought, temperature, metal, chemical, pathogenic and oxidativestresses, or combinations thereof, preferably drought and/ortemperature.

The term “expression” refers to the transcription and/or translation ofa codogenic gene segment or gene. As a rule, the resulting product is anmRNA or a protein. However, expression products can also includefunctional RNAs such as, for example, antisense, nucleic acids, tRNAs,snRNAs, rRNAs, RNAi, siRNA, ribozymes etc. Expression may be systemic,local or temporal, for example limited to certain cell types, tissue,sorgans or time periods.

Unless otherwise specified, the terms “polynucleotides”, “nucleic acid”and “nucleic acid molecule” are interchangeably in the present context.Unless otherwise specified, the terms “peptide”, “polypeptide” and“protein” are interchangeably in the present context. The term“sequence” may relate to polynucleotides, nucleic acids, nucleic acidmolecules, peptides, polypeptides and proteins, depending on the contextin which the term “sequence” is used. The terms “gene(s)”,“polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid molecule(s)” as used herein refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. The terms refer only to the primary structure ofthe molecule.

Thus, the terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”,“nucleotide sequence”, or “nucleic acid molecule(s)” as used hereininclude double- and single-stranded DNA and RNA. They also include knowntypes of modifications, for example, methylation, “caps”, substitutionsof one or more of the naturally occurring nucleotides with an analog.Preferably, the DNA or RNA sequence of the invention comprises a codingsequence encoding the herein defined polypeptide.

A “coding sequence” is a nucleotide sequence, which is transcribed intomRNA and/or translated into a polypeptide when placed under the controlof appropriate regulatory sequences. The boundaries of the codingsequence are determined by a translation start codon at the 5′-terminusand a translation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to mRNA, cDNA, recombinant nucleotidesequences or genomic DNA, while introns may be present as well undercertain circumstances.

For the purposes of the invention, as a rule the plural is intended toencompass the singular and vice versa.

Further, the transgenic plant cell is derived from a monocotyledonousplant. Alternatively, the transgenic plant cell is derived from adicotyledonous plant. Preferably, the transgenic plant cell is selectedfrom the group consisting of maize, wheat, rye, oat, triticale, rice,barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper,sunflower, flax, borage, sufflower, linseed, primrose, rapeseed, turniprape, tagetes, solanaceous plants, potato, tabacco, eggplant, tomato,Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oilpalm, coconut, perennial grass, forage crops and Arabidopsis thaliana.Moreover, the transgenic plant cell of the present invention can bederived from a gymnosperm plant. Preferably, the plant is selected fromthe group of spruce, pine and fir.

The invention further provides a seed produced by a transgenic planttransformed by a SRP coding nucleic acid, wherein the plant is truebreeding for increased tolerance to environmental stress, which ispreferably achieved by altering metabolic activity, as compared to awild type plant cell. The transgenic plant might be a monocot, a dicotor a gymnosperm plant. The invention further provides a seed produced bya transgenic plant expressing an SRP wherein the plant is true breedingfor increased tolerance to environmental stress, which is preferablyachieved by altering metabolic activity, as compared to a wild typeplant cell. The invention pertains to a seed produced by a transgenicplant, wherein the seed is genetically homozygous for a transgeneconferring an increased tolerance to environmental stress, which ispreferably achieved by altering metabolic activity, as compared to awild type plant.

The invention further provides an agricultural product produced by anyof the below-described transgenic plants, plant parts such as leafs,petal, anther, roots, tubers, stems, buds, flowers or seeds. Theinvention further provides a isolated recombinant expression vectorcomprising a SRP encoding nucleic acid.

The invention further provides a method of producing a transgenic plantwith a SRP coding nucleic acid, wherein expression of the nucleic acidin the plant results in increased tolerance and/or resistance to anenvironmental stress, which is preferably achieved by altering metabolicactivity, as compared to a corresponding non-transformed wild type plantcell, comprising

-   -   a) transforming a plant cell with an expression vector including        a SRP encoding nucleic acid selected from the group comprising        the nucleic acid of FIGS. 1 a, 1 b or 1 c and/or homologs or        parts thereof and    -   b) generating from the plant cell a transgenic plant with an        increased tolerance to environmental stress as compared to a        corresponding non-transformed wild type plant.

With regard to invention described here, “transformed or transgene”means all those plants or parts thereof which have been brought about bygenetic manipulation methods and in which either

-   -   c) one or more genes, preferably encoded by one or more nucleic        acid sequences as depicted in FIGS. 1 a, 1 b or 1 c and/or a        homolog thereof, or    -   d) a genetic regulatory element, for example a promoter, which        is functionally linked e.g. to the nucleic acid sequence as        depicted in FIGS. 1 a, 1 b or 1 c and/or a homolog thereof, or    -   e) (a) and (b)        is/are not present in its/their natural genetic environment or        has/have been modified by means of genetic manipulation methods,        it being possible for the modification to be, by way of example,        a substitution, addition, deletion, inversion or insertion of        one or more nucleotide radicals.

“Natural genetic environment” means the natural chromosomal locus in theorganism of origin or the presence in a genomic library. In the case ofa genomic library, the natural, genetic environment of the nucleic acidsequence is preferably at least partially still preserved. Theenvironment flanks the nucleic acid sequence at least on one side andhas a sequence length of at least 50 bp, preferably at least 500 bp,particularly preferably at least 1000 bp, very particularly preferablyat least 5000 bp.

In said method for producing a transgenic plant comprising an SRP, theSRP coding nucleic acid is selected from the group comprising thenucleic acid of FIGS. 1 a, 1 b or 1 c and/or homologs of the aforementioned sequences. Further, the SRP coding nucleic acid used in thesaid method is at least about 50% homologous to one of the nucleic acidof FIGS. 1 a, 1 b or 1 c.

A plant or plant cell is considered “true breeding” for a particulartrait if it is genetically homozygous for that trait to the extent that,when the true-breeding plant is self-pollinated, a significant amount ofindependent segregation of the trait among the progeny is not observed.In the present invention, the trait arises from the transgenicexpression of one or more DNA sequences introduced into a plant cell orplant.

The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the level of expression of aSRP nucleic acid in the plant. The invention provides one method ofproducing a transgenic plant with a synthetic, novel or modifiedtranscription factor that acts by increasing the transcription of a SRPgene. Theoretically it is also possible to obtain a decrease inexpression of the gene.

A method of detecting environmental stress in plant cells or plantscomprising screening the plant cells for altered metabolic activity ascompared to non-stress conditions is also in the scope of the invention.

Further a method of screening plant cells or plants for increasedtolerance and/or resistance to environmental stress comprising screeningthe plant cells under stress conditions for altered metabolic activityas compared to non-stress conditions is encompassed in the invention.

The present invention also encloses a method of breeding plant cells orplants towards increased tolerance and/or resistance to environmentalstress comprising screening the plant cells under stress conditions foraltered metabolic activity as compared to non-stress conditions andselecting those with increased tolerance and/or resistance toenvironmental stress.

In these methods metabolite activity is preferably altered concerningthe above metabolites and groups of metabolites.

The present invention also encompasses the use of altered metabolicactivity and/or a SRP encoding nucleic acid selected from the groupcomprising the nucleic acid of FIGS. 1 a, 1 b or 1 c and/or homologs ofthe afore mentioned sequences or parts thereof as markers for selectionof plants or plant cells with increased tolerance to environmentalstress.

The present invention further encompasses the use of altered metabolicactivity and/or a SRP encoding nucleic acid selected from the groupcomprising the nucleic acid of FIGS. 1 a, 1 b or 1 c and/or homologs ofthe afore mentioned sequences or parts thereof as markers for detectionof stress in plants or plant cells.

The present invention also provides methods of modifying stresstolerance of a crop plant comprising utilizing a SRP coding nucleic acidsequence to identify individual plants in populations segregating foreither increased or decreased environmental stress tolerance (DNAmarker).

In the said method of modifying stress tolerance of a plant the SRPencoding nucleic acid may be selected from the group comprising thenucleic acid of FIGS. 1 a, 1 b or 1 c and/or homologs of the aforementioned sequences. Further the SRP coding nucleic acid used thereinmay at least about 50% homologous to one of the nucleic acid of FIGS. 1a, 1 b or 1 c. Also an expression vector as described in the presentinvention might be used in the said method.

In a variant method of said method of modifying stress tolerance, theplant is transformed with an inducible promoter that directs expressionof the SRP. For example, the promoter is tissue specific. In a variantmethod, the used promoter is developmentally regulated.

In a further embodiment, the method of modifying stress tolerancecomprises one or more of the following steps:

-   -   a) stabilizing a protein conferring the increased expression of        a protein encoded by the nucleic acid molecule of the invention        or of the polypeptide of the invention having the        herein-mentioned activity of altering the metabolic activity;    -   b) stabilizing a mRNA conferring the increased expression of a        protein encoded by the nucleic acid molecule of the invention or        its homologs or of a mRNA encoding the polypeptide of the        present invention having the herein-mentioned activity of        altering the metabolic activity;    -   c) increasing the specific activity of a protein conferring the        increased expression of a protein encoded by the nucleic acid        molecule of the invention or of the polypeptide of the present        invention or decreasing the inhibitory regulation of the        polypeptide of the invention;    -   d) generating or increasing the expression of an endogenous or        artificial transcription factor mediating the expression of a        protein conferring the increased expression of a protein encoded        by the nucleic acid molecule of the invention or of the        polypeptide of the invention having the herein-mentioned        activity of altering the metabolic activity;    -   e) stimulating activity of a protein conferring the increased        expression of a protein encoded by the nucleic acid molecule of        the present invention or a polypeptide of the present invention        having the herein-mentioned activity of altering the metabolic        activity by adding one or more exogenous inducing factors to the        organisms or parts thereof;    -   f) expressing a transgenic gene encoding a protein conferring        the increased expression of a polypeptide encoded by the nucleic        acid molecule of the present invention or a polypeptide of the        present invention, having the herein-mentioned activity of        altering the metabolic activity; and/or    -   g) increasing the copy number of a gene conferring the increased        expression of a nucleic acid molecule encoding a polypeptide        encoded by the nucleic acid molecule of the invention or the        polypeptide of the invention having the herein-mentioned        activity of altering the metabolic activity;    -   h) increasing the expression of the endogenous gene encoding the        polypeptide of the invention or its homologs by adding positive        expression or removing negative expression elements, e.g.        homologous recombination can be used to either introduce        positive regulatory elements like for plants the 35S enhancer        into the promoter or to remove repressor elements form        regulatory regions. Further gene conversion methods can be used        to disrupt repressor elements or to enhance to activity of        positive elements-positive elements can be randomly introduced        in plants by T-DNA or transposon mutagenesis and lines can be        identified in which the positive elements have be integrated        near to a gene of the invention, the expression of which is        thereby enhanced; and/or    -   i) modulating growth conditions of the plant in such a manner,        that the expression or activity of the gene encoding the protein        of the invention or the protein itself is enhanced;    -   j) selecting of organisms with especially high activity of the        proteins of the invention from natural or from mutagenized        resources and breeding them into the target organisms, eg the        elite crops.

Preferably, said mRNA is the nucleic acid molecule of the presentinvention and/or the protein conferring the increased expression of aprotein encoded by the nucleic acid molecule of the present invention orthe polypeptide having the herein mentioned activity is the polypeptideof the present invention, e.g. conferring increased tolerance toenvironmental stress by altering the metabolic activity.

In general, the amount of mRNA, polynucleotide or nucleic acid moleculein a cell or a compartment of an organism correlates with the amount ofencoded protein and thus with the overall activity of the encodedprotein in said volume. Said correlation is not always linear, theactivity in the volume is dependent on the stability of the molecules,the degradation of the molecules or the presence of activating orinhibiting co-factors. Further, product and educt inhibitions of enzymesare well known, e.g. Zinser et al. “Enzyminhibitoren/Enzyme inhibitors”.

The activity of the abovementioned proteins and/or poylpeptide encodedby the nucleic acid molecule of the present invention can be increasedin various ways. For example, the activity in an organism or in a partthereof, like a cell, is increased via increasing the gene productnumber, e.g. by increasing the expression rate, like introducing astronger promoter, or by increasing the stability of the mRNA expressed,thus increasing the translation rate, and/or increasing the stability ofthe gene product, thus reducing the proteins decayed. Further, theactivity or turnover of enzymes can be influenced in such a way that areduction or increase of the reaction rate or a modification (reductionor increase) of the affinity to the substrate results, is reached. Amutation in the catalytic centre of an polypeptide of the invention,e.g. as enzyme, can modulate the turn over rate of the enzyme, e.g. aknock out of an essential amino acid can lead to a reduced or completelyknock out activity of the enzyme, or the deletion or mutation ofregulator binding sites can reduce a negative regulation like a feedbackinhibition (or a substrate inhibition, if the substrate level is alsoincreased). The specific activity of an enzyme of the present inventioncan be increased such that the turn over rate is increased or thebinding of a co-factor is improved. Improving the stability of theencoding mRNA or the protein can also increase the activity of a geneproduct. The stimulation of the activity is also under the scope of theterm “increased activity”.

Moreover, the regulation of the abovementioned nucleic acid sequencesmay be modified so that gene expression is increased. This can beachieved advantageously by means of heterologous regulatory sequences orby modifying, for example mutating, the natural regulatory sequenceswhich are present. The advantageous methods may also be combined witheach other.

In general, an activity of a gene product in an organism or partthereof, in particular in a plant cell, a plant, or a plant tissue or apart thereof or in a microorganism can be increased by increasing theamount of the specific encoding mRNA or the corresponding protein insaid organism or part thereof. “Amount of protein or mRNA” is understoodas meaning the molecule number of polypeptides or mRNA molecules in anorganism, a tissue, a cell or a cell compartment. “Increase” in theamount of a protein means the quantitative increase of the moleculenumber of said protein in an organism, a tissue, a cell or a cellcompartment or part thereof—for example by one of the methods describedherein below—in comparison to a wild type, control or reference.

The increase in molecule number amounts preferably to at least 1%,preferably to more than 10%, more preferably to 30% or more, especiallypreferably to 50%, 70% or more, very especially preferably to 100%, mostpreferably to 500% or more. However, a de novo expression is alsoregarded as subject of the present invention.

A modification, i.e. an increase or decrease, can be caused byendogenous or exogenous factors. For example, an increase in activity inan organism or a part thereof can be caused by adding a gene product ora precursor or an activator or an agonist to the media or nutrition orcan be caused by introducing said subjects into a organism, transient orstable.

In one embodiment the increase or decrease in metabolic activity in theplant or a part thereof, e.g. in a cell, a tissue, a organ, an organelleetc., is achieved by increasing the endogenous level of the polypeptideof the invention. Accordingly, in an embodiment of the presentinvention, the present invention relates to a process wherein the genecopy number of a gene encoding the polynucleotide or nucleic acidmolecule of the invention is increased. Further, the endogenous level ofthe polypeptide of the invention can for example be increased bymodifying the transcriptional or translational regulation of thepolypeptide.

In one embodiment the metabolic activity in the plant or part thereofcan be altered by targeted or random mutagenesis of the endogenous genesof the invention. For example homologous recombination can be used toeither introduce positive regulatory elements like for plants the 35Senhancer into the promoter or to remove repressor elements formregulatory regions. In addition gene conversion like methods describedby Kochevenko and Willmitzer (Plant Physiol. 2003 May; 132(1):174-84)and citations therein can be used to disrupt repressor elements or toenhance to activity of positive regulatory elements.

Furthermore positive elements can be randomly introduced in (plant)genomes by T-DNA or transposon mutagenesis and lines can be screenedfor, in which the positive elements has be integrated near to a gene ofthe invention, the expression of which is thereby enhanced. Theactivation of plant genes by random integrations of enhancer elementshas been described by Hayashi et al., 1992 (Science 258:1350-1353) orWeigel et al., 2000 (Plant Physiol. 122, 1003-1013) and others citatedtherein.

Reverse genetic strategies to identify insertions (which eventuallycarrying the activation elements) near in genes of interest have beendescribed for various cases eg. Krysan et al., 1999 (Plant Cell 1999,11, 2283-2290); Sessions et al., 2002 (Plant Cell 2002, 14, 2985-2994);Young et al., 2001, (Plant Physiol. 2001, 125, 513-518); Koprek et al.,2000 (Plant J. 2000, 24, 253-263); Jeon et al., 2000 (Plant J. 2000, 22,561-570); Tissier et al., 1999 (Plant Cell 1999, 11, 1841-1852);Speulmann et al., 1999 (Plant Cell 1999,11, 1853-1866). Briefly materialfrom all plants of a large T-DNA or transposon mutagenized plantpopulation is harvested and genomic DNA prepared. Then the genomic DNAis pooled following specific architectures as described for example inKrysan et al., 1999 (Plant Cell 1999, 11, 2283-2290). Pools of genomicsDNAs are then screened by specific multiplex PCR reactions detecting thecombination of the insertional mutagen (eg T-DNA or Transposon) and thegene of interest. Therefore PCR reactions are run on the DNA pools withspecific combinations of T-DNA or transposon border primers and genespecific primers. General rules for primer design can again be takenfrom Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290) Rescreening oflower levels DNA pools lead to the identification of individual plantsin which the gene of interest is activated by the insertional mutagen.

The enhancement of positive regulatory elements or the disruption orweaking of negative regulatory elements can also be achieved throughcommon mutagenesis techniques: The production of chemically or radiationmutated populations is a common technique and known to the skilledworker. Methods for plants are described by Koorneef et al. 1982 and thecitations therein and by Lightner and Caspar in “Methods in MolecularBiology” Vol 82. These techniques usually induce point mutations thatcan be identified in any known gene using methods such as TILLING(Colbert et al. 2001).

Accordingly, the expression level can be increased if the endogenousgenes encoding a polypeptide conferring an increased expression of thepolypeptide of the present invention, in particular genes comprising thenucleic acid molecule of the present invention, are modified viahomologous recombination, Tilling approaches or gene conversion

Regulatory sequences can be operatively linked to the coding region ofan endogenous protein and control its transcription and translation orthe stability or decay of the encoding mRNA or the expressed protein. Inorder to modify and control the expression, promoter, UTRs, splicingsites, processing signals, polyadenylation sites, terminators,enhancers, repressors, post transcriptional or posttranslationalmodification sites can be changed, added or amended For example, theactivation of plant genes by random integrations of enhancer elementshas been described by Hayashi et al., 1992 (Science 258:1350-1353) orWeigel et al., 2000 (Plant Physiol. 122, 1003-1013) and others citatedtherein. For example, the expression level of the endogenous protein canbe modulated by replacing the endogenous promoter with a strongertransgenic promoter or by replacing the endogenous 3′UTR with a 3′UTR,which provides more stability without amending the coding region.Further, the transcriptional regulation can be modulated by introductionof a artificial transcription factor as described in the examples.Alternative promoters, terminators and UTRs are described below.

The activation of an endogenous polypeptide having above-mentionedactivity, e.g. conferring an increased tolerance to environmental stressafter altering the metabolic activity can also be increased byintroducing a synthetic transcription factor, which binds close to thecoding region of the protein of the invention encoding gene andactivates its transcription. A chimeric zinc finger protein can beconstrued, which comprises a specific DNA-binding domain and anactivation domain as e.g. the VP16 domain of Herpes Simplex virus. Thespecific binding domain can bind to the regulatory region of the proteincoding region. The expression of the chimeric transcription factor in aplant leads to a specific expression of the protein of the invention,see e.g. in WO01/52620, Oriz, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99,13290 or Guan, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, 13296.

In one further embodiment of the method according to the invention,plants are used in which one of the abovementioned genes, or one of theabovementioned nucleic acids, is mutated in a way that the activity ofthe encoded gene products is less influenced by cellular factors, or notat all, in comparison with the unmutated proteins. For example, wellknown regulation mechanism of enzymic activity are substrate inhibitionor feed back regulation mechanisms. Ways and techniques for theintroduction of substitutions, deletions and additions of one or morebases, nucleotides or amino acids of a corresponding sequence aredescribed herein below in the corresponding paragraphs and thereferences listed there, e.g. in Sambrook et al., Molecular Cloning,Cold Spring Habour, N.Y., 1989. The person skilled in the art will beable to identify regulation domains and binding sites of regulators bycomparing the sequence of the nucleic acid molecule of the presentinvention or the expression product thereof with the state of the art bycomputer software means which comprise algorithms for the identifying ofbinding sites and regulation domains or by introducing into a nucleicacid molecule or in a protein systematically mutations and assaying forthose mutations which will lead to an increased specify activity or anincreased activity per volume, in particular per cell.

It is therefore advantageously to express in a plant a nucleic acidmolecule of the invention or a polypeptide of the invention derived froma evolutionary distantly related organism, as e.g. using a prokaryoticgene in a eukaryotic host, as in these cases the regulation mechanism ofthe host cell may not weaken the activity (cellular or specific) of thegene or its expression product

The mutation is introduced in such a way that the production of theamino acids is not adversely affected.

Less influence on the regulation of a gene or its gene product isunderstood as meaning a reduced regulation of the enzymatic activityleading to an increased specific or cellular activity of the gene or itsproduct. An increase of the enzymatic activity is understood as meaningan enzymatic activity, which is increased by at least 10%,advantageously at least 20, 30 or 40%, especially advantageously by atleast 50, 60 or 70% in comparison with the starting organism.

The invention provides that the above methods can be performed such thatthe stress tolerance is increased. It is also possible to obtain adecrease in stress tolerance.

The invention is not limited to specific nucleic acids, specificpolypeptides, specific cell types, specific host cells, specificconditions or specific methods etc. as such, but may vary and numerousmodifications and variations therein will be apparent to those skilledin the art. It is also to be understood that the terminology used hereinis for the purpose of describing specific embodiments only and is notintended to be limiting.

The present invention also relates to isolated Stress Related Proteins(SRP) which are selected from the group comprising the proteins of FIGS.1 a, 1 b or 1 c and/or homologs thereof. Preferably, the isolated StressRelated Proteins (SRP) of the present invention are selected from yeastor E. coli. Further, the present invention is related to isolated StressRelated Protein (SRP) encoding nucleic acids selected from the groupcomprising the nucleic acid of FIGS. 1 a, 1 b or 1 c and/or homologsthereof. Here, preferably, an isolated Stress Related Protein (SRP)encoding nucleic acid encodes an SRP which is selected from yeast or E.coli.

The present invention provides stress related gene sequences selectedfrom the group consisting of the nucleic acid of FIGS. 1 a, 1 b or c ofyeast, preferably from Saccharomyces cerevisiae or E. coli.

Homologs of the aforementioned sequences can be isolated advantageouslyfrom yeast, fungi, viruses, algae, bacteria, such as Acetobacter(subgen. Acetobacter) aceti; Acidithiobacillus ferrooxidans;Acinetobacter sp.; Actinobacillus sp; Aeromonas salmonicida;Agrobacterium tumefaciens; Aquifex aeolicus; Arcanobacterium pyogenes;Aster yellows phytoplasma; Bacillus sp.; Bifidobacterium sp.; Borreliaburgdorferi; Brevibacterium linens; Brucella melitensis; Buchnera sp.;Butyrivibrio fibrisolvens; Campylobacter jejuni; Caulobacter crescentus;Chlamydia sp.; Chlamydophila sp.; Chlorobium limicola; Citrobacterrodentium; Clostridium sp.; Comamonas testosteroni; Corynebacterium sp.;Coxiella burnetii; Deinococcus radiodurans; Dichelobacter nodosus;Edwardsiella ictaluri; Enterobacter sp.; Erysipelothrix rhusiopathiae;Escherichia coli; Flavobacterium sp.; Francisella tularensis; Frankiasp. Cpl1; Fusobacterium nucleatum; Geobacillus stearothermophilus;Gluconobacter oxydans; Haemophilus sp.; Helicobacter pylori; Klebsiellapneumoniae; Lactobacillus sp.; Lactococcus lactis; Listeria sp.;Mannheimia haemolytica; Mesorhizobium loti; Methylophaga thalassica;Microcystis aeruginosa; Microscilla sp. PRE1; Moraxella sp. TA144;Mycobacterium sp.; Mycoplasma sp.; Neisseria sp.; Nitrosomonas sp.;Nostoc sp. PCC 7120; Novosphingobium aromaticivorans; Oenococcus oeni;Pantoea citrea; Pasteurella multocida; Pediococcus pentosaceus;Phormidium foveolarum; Phytoplasma sp.; Plectonema boryanum; Prevotellaruminicola; Propionibacterium sp.; Proteus vulgaris; Pseudomonas sp.;Ralstonia sp.; Rhizobium sp.; Rhodococcus equi; Rhodothermus marinus;Rickettsia sp.; Riemerella anatipestifer; Ruminococcus flavefaciens;Salmonella sp.; Selenomonas ruminantium; Serratia entomophila; Shigellasp.; Sinorhizobium meliloti; Staphylococcus sp.; Streptococcus sp.;Streptomyces sp.; Synechococcus sp.; Synechocystis sp. PCC 6803;Thermotoga maritima; Treponema sp.; Ureaplasma urealyticum; Vibriocholerae; Vibrio parahaemolyticus; Xylella fastidiosa; Yersinia sp.;Zymomonas mobilis, preferably Salmonella sp. or Escherichia coli orplants, preferably from yeasts such as from the genera Saccharomyces,Pichia, Candida, Hansenula, Torulopsis or Schizosaccharomyces or plantssuch as Arabidopsis thaliana, maize, wheat, rye, oat, triticale, rice,barley, soybean, peanut, cotton, borage, sufflower, linseed, primrose,rapeseed, canola and turnip rape, manihot, pepper, sunflower, tagetes,solanaceous plant such as potato, tobacco, eggplant and tomato, Viciaspecies, pea, alfalfa, bushy plants such as coffee, cacao, tea, Salixspecies, trees such as oil palm, coconut, perennial grass, such asryegrass and fescue, and forage crops, such as alfalfa and clover andfrom spruce, pine or fir for example. More preferably homologs ofaforementioned sequences can be isolated from Saccharomyces cerevisiae,E. coli or plants, preferably Brassica napus, Glycine max, or Oryzasativa.

The stress related proteins of the present invention are preferablyproduced by recombinant DNA techniques. For example, a nucleic acidmolecule encoding the protein is cloned into an expression vector, forexample in to a binary vector, the expression vector is introduced intoa host cell, for example the Arabidopsis thaliana wild type NASC N906 orany other plant cell as described in the examples see below, and thestress related protein is expressed in said host cell. Examples forbinary vectors are pBIN19, pBl1101, pBinAR, pGPTV, pCAMBIA, pBIB-HYG,pBecks, pGreen or pPZP (Hajukiewicz, P. et al., 1994, Plant Mol. Biol.,25: 989-994 and Hellens et al, Trends in Plant Science (2000) 5,446-451.).

Advantageously, the nucleic acid sequences according to the invention orthe gene construct together with at least one reporter gene are clonedinto an expression cassette, which is introduced into the organism via avector or directly into the genome. This reporter gene should allow easydetection via a growth, fluorescence, chemical, bioluminescence orresistance assay or via a photometric measurement. Examples of reportergenes which may be mentioned are antibiotic- or herbicide-resistancegenes, hydrolase genes, fluorescence protein genes, bioluminescencegenes, sugar or nucleotide metabolic genes or biosynthesis genes such asthe Ura3 gene, the IIv2 gene, the luciferase gene, the β-galactosidasegene, the gfp gene, the 2-desoxyglucose-6-phosphate phosphatase gene,the □β-glucuronidase gene, β-lactamase gene, the neomycinphosphotransferase gene, the hygromycin phosphotransferase gene or theBASTA (=gluphosinate-resistance) gene. These genes permit easymeasurement and quantification of the transcription activity and henceof the expression of the genes. In this way genome positions may beidentified which exhibit differing productivity.

In a preferred embodiment a nucleic acid contruct, for example anexpression cassette, comprises upstream, i.e. at the 5′ end of theencoding sequence, a promoter and downstream, i.e. at the 3′ end, apolyadenylation signal and optionally other regulatory elements whichare operably linked to the intervening, encoding sequence with thenucleic acid of FIGS. 1 a, 1 b or 1 c. By an operable linkage is meantthe sequential arrangement of promoter, encoding sequence, terminatorand optionally other regulatory elements in such a way that each of theregulatory elements can fulfill its function in the expression of theencoding sequence in due manner. The sequences preferred for operablelinkage are targeting sequences for ensuring subcellular localization inplastids. However, targeting sequences for ensuring subcellularlocalization in the mitochondrium, in the endoplasmic reticulum (=ER),in the nucleus, in oil corpuscles or other compartments may also beemployed as well as translation promoters such as the 5′ lead sequencein tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15 (1987),8693-8711).

A nucleic acid construct, for example an expression cassette may, forexample, contain a constitutive promoter or a tissue-specific promoter(preferably the USP or napin promoter) the gene to be expressed and theER retention signal. For the ER retention signal the KDEL amino acidsequence (lysine, aspartic acid, glutamic acid, leucine) or the KKXamino acid sequence (lysine-lysine-X-stop, wherein X means every otherknown amino acid) is preferably employed.

For expression in a prokaryotic or eukaryotic host organism, for examplea microorganism such as a fungus or a plant the expression cassette isadvantageously inserted into a vector such as by way of example aplasmid, a phage or other DNA which allows optimum expression of thegenes in the host organism.

Examples of suitable plasmids are: in E. coli pLG338, pACYC184, pBRseries such as e.g. pBR322, pUC series such as pUC18 or pUC19, M113 mpseries, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290,plN-III¹¹³-B1, λgt11 or pBdCl; in Streptomyces pIJ101, pIJ364, pIJ702 orpIJ361; in Bacillus pUB110, pC194 or pBD214; in Corynebacterium pSA77 orpAJ667; in fungi pALS1, pIL2 or pBB116; other advantageous fungalvectors are described by Romanos, M. A. et al., [(1992), “Foreign geneexpression in yeast: a review”, Yeast 8: 423-488] and by van den Hondel,C. A. M. J. J. et al. [(1991) “Heterologous gene expression infilamentous fungi” as well as in More Gene Manipulations in Fungi [J. W.Bennet & L. L. Lasure, eds., pp. 396-428: Academic Press: San Diego] andin “Gene transfer systems and vector development for filamentous fungi”[van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) in: AppliedMolecular Genetics of Fungi, Peberdy, J. F. et al., eds., pp. 1-28,Cambridge University Press: Cambridge]. Examples of advantageous yeastpromoters are 2 μM, pAG-1, YEp6, YEp13 or pEMBLYe23. Examples of algalor plant promoters are pLGV23, pGHlac⁺, pBIN19, pAK2004, pVKH or pDH51(see Schmidt, R. and Willmitzer, L., 1988). The vectors identified aboveor derivatives of the vectors identified above are a small selection ofthe possible plasmids. Further plasmids are well known to those skilledin the art and may be found, for example, in the book Cloning Vectors(Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985ISBN 0 444 904018). Suitable plant vectors are described inter alia in“Methods in Plant Molecular Biology and Biotechnology” (CRC Press), Ch.6/7, pp. 71-119. Advantageous vectors are known as shuttle vectors orbinary vectors which replicate in E. coli and Agrobacterium.

By vectors is meant with the exception of plasmids all other vectorsknown to those skilled in the art such as by way of example phages,viruses such as SV40, CMV, baculovirus, adenovirus, transposons, ISelements, phasmids, phagemids, cosmids, linear or circular DNA. Thesevectors can be replicated autonomously in the host organism or bechromosomally replicated, chromosomal replication being preferred.

In a further embodiment of the vector the expression cassette accordingto the invention may also advantageously be introduced into theorganisms in the form of a linear DNA and be integrated into the genomeof the host organism by way of heterologous or homologous recombination.This linear DNA may be composed of a linearized plasmid or only of theexpression cassette as vector or the nucleic acid sequences according tothe invention.

In a further advantageous embodiment the nucleic acid sequence accordingto the invention can also be introduced into an organism on its own.

If in addition to the nucleic acid sequence according to the inventionfurther genes are to be introduced into the organism, all together witha reporter gene in a single vector or each single gene with a reportergene in a vector in each case can be introduced into the organism,whereby the different vectors can be introduced simultaneously orsuccessively.

The vector advantageously contains at least one copy of the nucleic acidsequences according to the invention and/or the expression cassette(=gene construct) according to the invention.

The invention further provides an isolated recombinant expression vectorcomprising a SRP nucleic acid as described above, wherein expression ofthe vector in a host cell results in increased tolerance toenvironmental stress as compared to a wild type variety of the hostcell. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors.” In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses, and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. As used herein with respect to arecombinant expression vector, “operatively linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers, andother expression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) and Gruber and Crosby, in: Methods in PlantMolecular Biology and Biotechnology, eds. Glick and Thompson, Chapter 7,89-108, CRC Press: Boca Raton, Fla., including the references therein.Regulatory sequences include those that direct constitutive expressionof a nucleotide sequence in many types of host cells and those thatdirect expression of the nucleotide sequence only in certain host cellsor under certain conditions. It will be appreciated by those skilled inthe art that the design of the expression vector can depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of polypeptide desired, etc. The expression vectors of theinvention can be introduced into host cells to thereby producepolypeptides or peptides, including fusion polypeptides or peptides,encoded by nucleic acids as described herein (e.g., SRPs, mutant formsof SRPs, fusion polypeptides, etc.).

The recombinant expression vectors of the invention can be designed forexpression of SRPs in prokaryotic or eukaryotic cells. For example, SRPgenes can be expressed in bacterial cells such as C. glutamicum, insectcells (using baculovirus expression vectors), yeast and other fungalcells (See Romanos, M. A. et al., 1992, Foreign gene expression inyeast: a review, Yeast 8:423-488; van den Hondel, C. A. M. J. J. et al.,1991, Heterologous gene expression in filamentous fungi, in: More GeneManipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p. 396-428:Academic Press: San Diego; and van den Hondel, C. A. M. J. J. & Punt, P.J., 1991, Gene transfer systems and vector development for filamentousfungi, in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al.,eds., p. 1-28, Cambridge University Press: Cambridge), algae (Falciatoreet al., 1999, Marine Biotechnology 1(3):239-251), ciliates of the types:Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena,Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae with vectors following a transformationmethod as described in PCT Application No. WO 98/01572, andmulticellular plant cells (See Schmidt, R. and Willmitzer, L., 1988,High efficiency Agrobacterium tumefaciens-mediated transformation ofArabidopsis thaliana leaf and cotyledon explants, Plant Cell Rep.583-586; Plant Molecular Biology and Biotechnology, C Press, Boca Raton,Fla., chapter 6/7, S.71-119 (1993); F. F. White, B. Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds. Kung und R. Wu, 128-43, Academic Press: 1993;Potrykus, 1991, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42:205-225and references cited therein) or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press: San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of polypeptides in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion polypeptides. Fusion vectorsadd a number of amino acids to a polypeptide encoded therein, usually tothe amino terminus of the recombinant polypeptide but also to theC-terminus or fused within suitable regions in the polypeptides. Suchfusion vectors typically serve three purposes: 1) to increase expressionof a recombinant polypeptide; 2) to increase the solubility of arecombinant polypeptide; and 3) to aid in the purification of arecombinant polypeptide by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantpolypeptide to enable separation of the recombinant polypeptide from thefusion moiety subsequent to purification of the fusion polypeptide. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin, and enterokinase.

By way of example the plant expression cassette can be installed in thepRT transformation vector ((a) Toepfer et al., 1993, Methods Enzymol.,217: 66-78; (b) Toepfer et al. 1987, Nucl. Acids. Res. 15: 5890 ff.).

Alternatively, a recombinant vector (=expression vector) can also betranscribed and translated in vitro, e.g. by using the T7 promoter andthe T7 RNA polymerase.

Expression vectors employed in prokaryotes frequently make use ofinducible systems with and without fusion proteins or fusionoligopeptides, wherein these fusions can ensue in both N-terminal andC-terminal manner or in other useful domains of a protein. Such fusionvectors usually have the following purposes: i.) to increase the RNAexpression rate; ii.) to increase the achievable protein synthesis rate;iii.) to increase the solubility of the protein; iv.) or to simplifypurification by means of a binding sequence usable for affinitychromatography. Proteolytic cleavage points are also frequentlyintroduced via fusion proteins, which allow cleavage of a portion of thefusion protein and purification. Such recognition sequences forproteases are recognized, e.g. factor Xa, thrombin and enterokinase.

Typical advantageous fusion and expression vectors are pGEX [PharmaciaBiotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67: 31-40],pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which contains glutathione S-transferase (GST),maltose binding protein or protein A.

In one embodiment, the coding sequence of the SRP is cloned into a pGEXexpression vector to create a vector encoding a fusion polypeptidecomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X polypeptide. The fusion polypeptide can be purified by affinitychromatography using glutathione-agarose resin. Recombinant PKSRPunfused to GST can be recovered by cleavage of the fusion polypeptidewith thrombin.

Other examples of E. coli expression vectors are pTrc [Amann et al.,(1988) Gene 69:301-315] and pET vectors [Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 60-89; Stratagene, Amsterdam, The Netherlands].

Target gene expression from the pTrc vector relies on host RNApolymerase transcription from a hybrid trp-lac fusion promoter. Targetgene expression from the pET 11d vector relies on transcription from aT7 gn10-lac fusion promoter mediated by a co-expressed viral RNApolymerase (T7 gn1). This viral polymerase is supplied by host strainsBL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gn1gene under the transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant polypeptide expression is toexpress the polypeptide in a host bacteria with an impaired capacity toproteolytically cleave the recombinant polypeptide (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the sequenceof the nucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin the bacterium chosen for expression, such as C. glutamicum (Wada etal., 1992, Nucleic Acids Res. 20:2111-2118). Such alteration of nucleicacid sequences of the invention can be carried out by standard DNAsynthesis techniques.

Other advantageous vectors for use in yeast are pYepSec1 (Baldari, etal., (1987) Embo J. 6:229-234), pMFα(Kurjan and Herskowitz, (1982) Cell30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYESderivatives (Invitrogen Corporation, San Diego, Calif.). Vectors for usein filamentous fungi are described in: van den Hondel, C. A. M. J. J. &Punt, P. J. (1991) “Gene transfer systems and vector development forfilamentous fungi”, in: Applied Molecular Genetics of Fungi, J. F.Peberdy, et al., eds., pp. 1-28, Cambridge University Press: Cambridge.

Alternatively, insect cell expression vectors can also be advantageouslyutilized, e.g. for expression in Sf 9 cells. These are e.g. the vectorsof the pAc series (Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) andthe pVL series (Lucklow and Summers (1989) Virology 170:31-39).

Furthermore, plant cells or algal cells can advantageously be used forgene expression. Examples of plant expression vectors may be found inBecker, D., et al. (1992) “New plant binary vectors with selectablemarkers located proximal to the left border”, Plant Mol. Biol. 20:1195-1197 or in Bevan, M. W. (1984), “Binary Agrobacterium vectors forplant transformation”, Nucl. Acid. Res. 12: 8711-8721.

Furthermore, the nucleic acid sequences may also be expressed inmammalian cells, advantageously in nonhuman mammalian cells. Examples ofcorresponding expression vectors are pCDM8 and pMT2PC referred to in:Seed, B. (1987) Nature 329:840 or Kaufman et al. (1987) EMBO J. 6:187-195). At the same time promoters preferred for use are of viralorigin, such as by way of example promoters of polyoma, adenovirus 2,cytomegalovirus or simian virus 40. Other prokaryotic and eukaryoticexpression systems are referred to in chapters 16 and 17 of Sambrook etal., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

In a preferred embodiment of the present invention, the SRPs areexpressed in plants and plants cells such as unicellular plant cells(e.g. algae) (See Falciatore et al., 1999, Marine Biotechnology1(3):239-251 and references therein) and plant cells from higher plants(e.g., the spermatophytes, such as crop plants). A SRP may be“introduced” into a plant cell by any means, including transfection,transformation or transduction, electroporation, particle bombardment,agroinfection, and the like. One transformation method known to those ofskill in the art is the dipping of a flowering plant into anAgrobacteria solution, wherein the Agrobacteria contains the SRP nucleicacid, followed by breeding of the transformed gametes.

Other suitable methods for transforming or transfecting host cellsincluding plant cells can be found in Sambrook, et al., MolecularCloning: A Laboratory Manual. 2^(nd), ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, and other laboratory manuals such as Methods in MolecularBiology, 1995, Vol. 44, Agrobacterium protocols, ed: Gartland and Davey,Humana Press, Totowa, N.J. As biotic and abiotic stress tolerance is ageneral trait wished to be inherited into a wide variety of plants likemaize, wheat, rye, oat, triticale, rice, barley, soybean, peanut,cotton, rapeseed and canola, manihot, pepper, sunflower and tagetes,solanaceous plants like potato, tobacco, eggplant, and tomato, Viciaspecies, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species,trees (oil palm, coconut), perennial grasses, and forage crops, thesecrop plants are also preferred target plants for a genetic engineeringas one further embodiment of the present invention. Forage cropsinclude, but are not limited to, Wheatgrass, Canarygrass, Bromegrass,Wildrye Grass, Bluegrass, Orchardgrass, Alfalfa, Salfoin, BirdsfootTrefoil, Alsike Clover, Red Clover, and Sweet Clover.

In one embodiment of the present invention, transfection of a SRP into aplant is achieved by Agrobacterium mediated gene transfer. Agrobacteriummediated plant transformation can be performed using for example theGV3101 (pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396) orLBA4404 (Clontech) Agrobacterium tumefaciens strain. Transformation canbe performed by standard transformation and regeneration techniques(Deblaere et al., 1994, Nucl. Acids Res. 13:4777-4788; Gelvin, StantonB. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2^(nd)Ed.—Dordrecht: Kluwer Academic Publ., 1995.—in Sect., Ringbuc ZentraleSignatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R.; Thompson, JohnE., Methods in Plant Molecular Biology and Biotechnology, Boca Raton:CRC Press, 1993 360 S., ISBN 0-8493-5164-2). For example, rapeseed canbe transformed via cotyledon or hypocotyl transformation (Moloney etal., 1989, Plant cell Report 8:238-242; De Block et al., 1989, PlantPhysiol. 91:694-701). Use of antibiotics for Agrobacterium and plantselection depends on the binary vector and the Agrobacterium strain usedfor transformation. Rapeseed selection is normally performed usingkanamycin as selectable plant marker. Agrobacterium mediated genetransfer to flax can be performed using, for example, a techniquedescribed by Mlynarova et al., 1994, Plant Cell Report 13:282-285.Additionally, transformation of soybean can be performed using forexample a technique described in European Patent No. 0424 047, U.S. Pat.No. 5,322,783, European Patent No. 0397 687, U.S. Pat. No. 5,376,543, orU.S. Pat. No. 5,169,770. Transformation of maize can be achieved byparticle bombardment, polyethylene glycol mediated DNA uptake or via thesilicon carbide fiber technique. (See, for example, Freeling and Walbot“The maize handbook” Springer Verlag: New York (1993) ISBN3-540-97826-7). A specific example of maize transformation is found inU.S. Pat. No. 5,990,387, and a specific example of wheat transformationcan be found in PCT Application No. WO 93/07256.

According to the present invention, the introduced SRP may be maintainedin the plant cell stably if it is incorporated into a non-chromosomalautonomous replicon or integrated into the plant chromosomes.Alternatively, the introduced SRP may be present on an extra-chromosomalnon-replicating vector and be transiently expressed or transientlyactive.

In one embodiment, a homologous recombinant microorganism can be createdwherein the SRP is integrated into a chromosome, a vector is preparedwhich contains at least a portion of a SRP gene into which a deletion,addition, or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the SRP gene.

Preferably, the SRP gene is a yeast, E. coli, Brassica napus, Glycinemax, or Oryza sativa SRP gene, but it can be a homolog from a relatedplant or even from a mammalian or insect source. In one embodiment, thevector is designed such that, upon homologous recombination, theendogenous SRP gene is functionally disrupted (i.e., no longer encodes afunctional polypeptide; also referred to as a knock-out vector).Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous SRP gene is mutated or otherwise alteredbut still encodes a functional polypeptide (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous SRP). To create a point mutation via homologousrecombination, DNA-RNA hybrids can be used in a technique known aschimeraplasty (Cole-Strauss et al., 1999, Nucleic Acids Research27(5):1323-1330 and Kmiec, 1999 Gene therapy American Scientist.87(3):240-247). Homologous recombination procedures in Physcomitrellapatens are also well known in the art and are contemplated for useherein.

Whereas in the homologous recombination vector, the altered portion ofthe SRP gene is flanked at its 5′ and 3′ ends by an additional nucleicacid molecule of the SRP gene to allow for homologous recombination tooccur between the exogenous SRP gene carried by the vector and anendogenous SRP gene, in a microorganism or plant. The additionalflanking SRP nucleic acid molecule is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several hundreds of base pairs up to kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector. See, e.g., Thomas, K.R., and Capecchi, M. R., 1987, Cell 51:503 for a description ofhomologous recombination vectors or Strepp et al., 1998, PNAS, 95(8):4368-4373 for cDNA based recombination in Physcomitrella patens).The vector is introduced into a microorganism or plant cell (e.g., viapolyethylene glycol mediated DNA), and cells in which the introducedPKSRP gene has homologously recombined with the endogenous PKSRP geneare selected using art-known techniques.

In another embodiment, recombinant microorganisms can be produced thatcontain selected systems which allow for regulated expression of theintroduced gene. For example, inclusion of a SRP gene on a vectorplacing it under control of the lac operon permits expression of the SRPgene only in the presence of IPTG. Such regulatory systems are wellknown in the art.

Whether present in an extra-chromosomal non-replicating vector or avector that is integrated into a chromosome, the SRP polynucleotidepreferably resides in a plant expression cassette. A plant expressioncassette preferably contains regulatory sequences capable of drivinggene expression in plant cells that are operatively linked so that eachsequence can fulfill its function, for example, termination oftranscription by polyadenylation signals. Preferred polyadenylationsignals are those originating from Agrobacterium tumefaciens t-DNA suchas the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5(Gielen et al., 1984, EMBO J. 3:835) or functional equivalents thereofbut also all other terminators functionally active in plants aresuitable. As plant gene expression is very often not limited ontranscriptional levels, a plant expression cassette preferably containsother operatively linked sequences like translational enhancers such asthe overdrive-sequence containing the 5′-untranslated leader sequencefrom tobacco mosaic virus enhancing the polypeptide per RNA ratio(Gallie et al., 1987, Nucl. Acids Research 15:8693-8711). Examples ofplant expression vectors include those detailed in: Becker, D. et al.,1992, New plant binary vectors with selectable markers located proximalto the left border, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W.,1984, Binary Agrobacterium vectors for plant transformation, Nucl. Acid.Res. 12:8711-8721; and Vectors for Gene Transfer in Higher Plants; in:Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung andR. Wu, Academic Press, 1993, S. 15-38.

“Transformation” is defined herein as a process for introducingheterologous DNA into a plant cell, plant tissue, or plant. It may occurunder natural or artificial conditions using various methods well knownin the art. Transformation may rely on any known method for theinsertion of foreign nucleic acid sequences into aprokaryotic oreukaryotic host cell. The method is selected based on the host cellbeing transformed and may include, but is not limited to, viralinfection, electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time. Transformed plant cells, plant tissue, or plants areunderstood to encompass not only the end product of a transformationprocess, but also transgenic progeny thereof.

The terms “transformed,” “transgenic,” and “recombinant” refer to a hostorganism such as a bacterium or a plant into which a heterologousnucleic acid molecule has been introduced. The nucleic acid molecule canbe stably integrated into the genome of the host or the nucleic acidmolecule can also be present as an extrachromosomal molecule. Such anextrachromosomal molecule can be auto-replicating. Transformed cells,tissues, or plants are understood to encompass not only the end productof a transformation process, but also transgenic progeny thereof. A“non-transformed,” “non-transgenic,” or “non-recombinant” host refers toa wild-type organism, e.g., a bacterium or plant, which does not containthe heterologous nucleic acid molecule.

A “transgenic plant”, as used herein, refers to a plant which contains aforeign nucleotide sequence inserted into either its nuclear genome ororganellar genome. It encompasses further the offspring generations i.e.the T1-, T2- and consecutively generations or BC1-, BC2- andconsecutively generation as well as crossbreeds thereof withnon-transgenic or other transgenic plants.

The host organism (=transgenic organism) advantageously contains atleast one copy of the nucleic acid according to the invention and/or ofthe nucleic acid construct according to the invention.

In principle all plants can be used as host organism. Preferredtransgenic plants are, for example, selected from the familiesAceraceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae,Cucurbitaceae, Euphorbiaceae, Fabaceae, Malvaceae, Nymphaeaceae,Papaveraceae, Rosaceae, Salicaceae, Solanaceae, Arecaceae, Bromeliaceae,Cyperaceae, Iridaceae, Liliaceae, Orchidaceae, Gentianaceae, Labiaceae,Magnoliaceae, Ranunculaceae, Carifolaceae, Rubiaceae, Scrophulariaceae,Caryophyllaceae, Ericaceae, Polygonaceae, Violaceae, Juncaceae orPoaceae and preferably from a plant selected from the group of thefamilies Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae,Papaveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae. Preferred arecrop plants such as plants advantageously selected from the group of thegenus peanut, oilseed rape, canola, sunflower, safflower, olive, sesame,hazelnut, almond, avocado, bay, pumpkin/squash, linseed, soya,pistachio, borage, maize, wheat, rye, oats, sorghum and millet,triticale, rice, barley, cassava, potato, sugarbeet, egg plant, alfalfa,and perennial grasses and forage plants, oil palm, vegetables(brassicas, root vegetables, tuber vegetables, pod vegetables, fruitingvegetables, onion vegetables, leafy vegetables and stem vegetables),buckwheat, Jerusalem artichoke, broad bean, vetches, lentil, dwarf bean,lupin, clover and Lucerne for mentioning only some of them.

In one preferred embodiment, the host plant is selected from thefamilies Aceraceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae,Cactaceae, Cucurbitaceae, Euphorbiaceae, Fabaceae, Malvaceae,Nymphaeaceae, Papaveraceae, Rosaceae, Salicaceae, Solanaceae, Arecaceae,Bromeliaceae, Cyperaceae, Iridaceae, Liliaceae, Orchidaceae,Gentianaceae, Labiaceae, Magnoliaceae, Ranunculaceae, Carifolaceae,Rubiaceae, Scrophulariaceae, Caryophyllaceae, Ericaceae, Polygonaceae,Violaceae, Juncaceae or Poaceae and preferably from a plant selectedfrom the group of the families Apiaceae, Asteraceae, Brassicaceae,Cucurbitaceae, Fabaceae, Papaveraceae, Rosaceae, Solanaceae, Liliaceaeor Poaceae. Preferred are crop plants and in particular plants mentionedherein above as host plants such as the families and genera mentionedabove for example preferred the species Anacardium occidentale,Calendula officinalis, Carthamus tinctorius, Cichorium intybus, Cynarascolymus, Helianthus annus, Tagetes lucida, Tagetes erecta, Tagetestenuifolia; Daucus carota; Corylus avellana, Corylus colurna, Boragoofficinalis; Brassica napus, Brassica rapa ssp., Sinapis arvensisBrassica juncea, Brassica juncea var. juncea, Brassica juncea var.crispifolia, Brassica juncea var. foliosa, Brassica nigra, Brassicasinapioides, Melanosinapis communis, Brassica oleracea, Arabidopsisthaliana, Anana comosus, Ananas ananas, Bromelia comosa, Carica papaya,Cannabis sative, Ipomoea batatus, Ipomoea pandurata, Convolvulusbatatas, Convolvulus tiliaceus, Ipomoea fastigiata, Ipomoea tiliacea,Ipomoea triloba, Convolvulus panduratus, Beta vulgaris, Beta vulgarisvar. altissima, Beta vulgaris var. vulgaris, Beta maritima, Betavulgaris var. perennis, Beta vulgaris var. conditiva, Beta vulgaris var.esculenta, Cucurbita maxima, Cucurbita mixta, Cucurbita pepo, Cucurbitamoschata, Olea europaea, Manihot utilissima, Janipha manihot, Jatrophamanihot, Manihot aipil, Manihot dulcis, Manihot manihot, Manihotmelanobasis, Manihot esculenta, Ricinus communis, Pisum sativum, Pisumarvense, Pisum humile, Medicago sativa, Medicago falcata, Medicagovaria, Glycine max Dolichos soja, Glycine gracilis, Glycine hispida,Phaseolus max, Soja hispida, Soja max, Cocos nucifera, Pelargoniumgrossularioides, Oleum cocoas, Laurus nobilis, Persea americana, Arachishypogaea, Linum usitatissimum, Linum humile, Linum austriacum, Linumbienne, Linum angustifolium, Linum catharticum, Linum flavum, Linumgrandiflorum, Adenolinum grandiflorum, Linum lewisii, Linum narbonense,Linum perenne, Linum perenne var. lewisii, Linum pratense, Linumtrigynum, Punica granatum, Gossypium hirsutum, Gossypium arboreum,Gossypium barbadense, Gossypium herbaceum, Gossypium thurberi, Musanana, Musa acuminata, Musa paradisiaca, Musa spp., Elaeis guineensis,Papaver orientale, Papaver rhoeas, Papaver dubium, Sesamum indicum,Piper aduncum, Piper amalago, Piper angustifolium, Piper auritum, Piperbetel, Piper cubeba, Piper longum, Piper nigrum, Piper retrofractum,Artanthe adunca, Artanthe elongata, Peperomia elongata, Piper elongatum,Steffensia elongata, Hordeum vulgare, Hordeum jubatum, Hordeum murinum,Hordeum secalinum, Hordeum distichon Hordeum aegiceras, Hordeumhexastichon., Hordeum hexastichum, Hordeum irregulare, Hordeum sativum,Hordeum secalinum, Avena sativa, Avena fatua, Avena byzantina, Avenafatua var. sativa, Avena hybrida, Sorghum bicolor, Sorghum halepense,Sorghum saccharatum, Sorghum vulgare, Andropogon drummondii, Holcusbicolor, Holcus sorghum, Sorghum aethiopicum, Sorghum arundinaceum,Sorghum caffrorum, Sorghum cernuum, Sorghum dochna, Sorghum drummondii,Sorghum durra, Sorghum guineense, Sorghum lanceolatum, Sorghum nervosum,Sorghum saccharatum, Sorghum subglabrescens, Sorghum verticilliflorum,Sorghum vulgare, Holcus halepensis, Sorghum miliaceum millet, Panicummilitaceum, Zea mays, Triticum aestivum, Triticum durum, Triticumturgidum, Triticum hybernum, Triticum macha, Triticum sativum orTriticum vulgare, Cofea spp., Coffea arabica, Coffea canephora, Coffealiberica, Capsicum annuum, Capsicum annuum var. glabriusculum, Capsicumfrutescens, Capsicum annuum, Nicotiana tabacum, Solanum tuberosum,Solanum melongena, Lycopersicon esculentum, Lycopersicon lycopersicum,Lycopersicon pyriforme, Solanum integrifolium, Solanum lycopersicumTheobroma cacao or Camellia sinensis.

Anacardiaceae such as the genera Pistacia, Mangifera, Anacardium e.g.the species Pistacia vera [pistachios, Pistazie], Mangifer indica[Mango] or Anacardium occidentale [Cashew]; Asteraceae such as thegenera Calendula, Carthamus, Centaurea, Cichorium, Cynara, Helianthus,Lactuca, Locusta, Tagetes, Valeriana e.g. the species Calendulaofficinalis [Marigold], Carthamus tinctorius [safflower], Centaureacyanus [cornflower], Cichorium intybus [blue daisy], Cynara scolymus[Artichoke], Helianthus annus [sunflower], Lactuca sativa, Lactucacrispa, Lactuca esculenta, Lactuca scariola L. ssp. sativa, Lactucascariola L. var. integrata, Lactuca scariola L. var. integrifolia,Lactuca sativa subsp. romana, Locusta communis, Valeriana locusta[lettuce], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia[Marigold]; Apiaceae such as the genera Daucus e.g. the species Daucuscarota [carrot]; Betulaceae such as the genera Corylus e.g. the speciesCorylus avellana or Corylus colurna [hazelnut]; Boraginaceae such as thegenera Borago e.g. the species Borago officinalis [borage]; Brassicaceaesuch as the genera Brassica, Melanosinapis, Sinapis, Arabadopsis e.g.the species Brassica napus, Brassica rapa ssp. [canola, oilseed rape,turnip rape], Sinapis arvensis Brassica juncea, Brassica juncea var.juncea, Brassica juncea var. crispifolia, Brassica juncea var. foliosa,Brassica nigra, Brassica sinapioides, Melanosinapis communis [mustard],Brassica oleracea [fodder beet] or Arabidopsis thaliana; Bromeliaceaesuch as the genera Anana, Bromelia e.g. the species Anana comosus,Ananas ananas or Bromelia comosa [pineapple]; Caricaceae such as thegenera Carica e.g. the species Carica papaya [papaya]; Cannabaceae suchas the genera Cannabis e.g. the species Cannabis sative [hemp],Convolvulaceae such as the genera Ipomea, Convolvulus e.g. the speciesIpomoea batatus, Ipomoea pandurata, Convolvulus batatas, Convolvulustiliaceus, Ipomoea fastigiata, pomoea tiliacea, Ipomoea triloba orConvolvulus panduratus [sweet potato, Man of the Earth, wild potato],Chenopodiaceae such as the genera Beta, i.e. the species Beta vulgaris,Beta vulgaris var. altissima, Beta vulgaris var. Vulgaris, Betamaritima, Beta vulgaris var. perennis, Beta vulgaris var. conditiva orBeta vulgaris var. esculenta [sugar beet]; Cucurbitaceae such as thegenera Cucubita e.g. the species Cucurbita maxima, Cucurbita mixta,Cucurbita pepo or Cucurbita moschata [pumpkin, squash]; Elaeagnaceaesuch as the genera Elaeagnus e.g. the species Olea europaea [olive];Ericaceae such as the genera Kalmia e.g. the species Kalmia latifolia,Kalmia angustifolia, Kalmia microphylla, Kalmia polifolia, Kalmiaoccidentalis, Cistus chamaerhodendros or Kalmia lucida [American laurel,broad-leafed laurel, calico bush, spoon wood, sheep laurel, alpinelaurel, bog laurel, western bog-laurel, swamp-laurel]; Euphorbiaceaesuch as the genera Manihot, Janipha, Jatropha, Ricinus e.g. the speciesManihot utilissima, Janipha manihot, Jatropha manihot, Manihot aipil,Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot esculenta[manihot, arrowroot, tapioca, cassava] or Ricinus communis [castor bean,Castor Oil Bush, Castor Oil Plant, Palma Christi, Wonder Tree]; Fabaceaesuch as the genera Pisum, Albizia, Cathormion, Feuillea, Inga,Pithecolobium, Acacia, Mimosa, Medicajo, Glycine, Dolichos, Phaseolus,Soja e.g. the species Pisum sativum, Pisum arvense, Pisum humile [pea],Albizia berteriana, Albizia julibrissin, Albizia lebbeck, Acaciaberteriana, Acacia littoralis, Albizia berteriana, Albizzia berteriana,Cathormion berteriana, Feuillea berteriana, Inga fragrans,Pithecellobium berterianum, Pithecellobium fragrans, Pithecolobiumberterianum, Pseudalbizzia berteriana, Acacia julibrissin, Acacia nemu,Albizia nemu, Feuilleea julibrissin, Mimosa julibrissin, Mimosaspeciosa, Sericanrda julibrissin, Acacia lebbeck, Acacia macrophylla,Albizia lebbek, Feuilleea lebbeck, Mimosa lebbeck, Mimosa speciosa[bastard logwood, silk tree, East Indian Walnut], Medicago sativa,Medicago falcata, Medicago varia [alfalfa]Glycine max Dolichos soja,Glycine gracilis, Glycine hispida, Phaseolus max, Soja hispida or Sojamax [soybean]; Geraniaceae such as the genera Pelargonium, Cocos, Oleume.g. the species Cocos nucifera, Pelargonium grossularioides or Oleumcocois [coconut]; Gramineae such as the genera Saccharum e.g. thespecies Saccharum officinarum; Juglandaceae such as the genera Juglans,Wallia e.g. the species Juglans regia, Juglans ailanthifolia, Juglanssieboldiana, Juglans cinerea, Wallia cinerea, Juglans bixbyi, Juglanscalifornica, Juglans hindsii, Juglans intermedia, Juglans jamaicensis,Juglans major, Juglans microcarpa, Juglans nigra or Wallia nigra[walnut, black walnut, common walnut, persian walnut, white walnut,butternut, black walnut]; Lauraceae such as the genera Persea, Lauruse.g. the species laurel Laurus nobilis [bay, laurel, bay laurel, sweetbay], Persea americana Persea americana, Persea gratissima or Perseapersea [avocado]; Leguminosae such as the genera Arachis e.g. thespecies Arachis hypogaea [peanut]; Linaceae such as the genera Linum,Adenolinum e.g. the species Linum usitatissimum, Linum humile, Linumaustriacum, Linum bienne, Linum angustifolium, Linum catharticum, Linumflavum, Linum grandiflorum, Adenolinum grandiflorum, Linum lewisii,Linum narbonense, Linum perenne, Linum perenne var. lewisii, Linumpratense or Linum trigynum [flax, linseed]; Lythrarieae such as thegenera Punica e.g. the species Punica granatum [pomegranate]; Malvaceaesuch as the genera Gossypium e.g. the species Gossypium hirsutum,Gossypium arboreum, Gossypium barbadense, Gossypium herbaceum orGossypium thurberi [cotton]; Musaceae such as the genera Musa e.g. thespecies Musa nana, Musa acuminata, Musa paradisiaca, Musa spp. [banana];Onagraceae such as the genera Camissonia, Oenothera e.g. the speciesOenothera biennis or Camissonia brevipes [primrose, evening primrose];Palmae such as the genera Elacis e.g. the species Elaeis guineensis [oilplam]; Papaveraceae such as the genera Papaver e.g. the species Papaverorientale, Papaver rhoeas, Papaver dubium [poppy, oriental poppy, cornpoppy, field poppy, shirley poppies, field poppy, long-headed poppy,long-pod poppy]; Pedaliaceae such as the genera Sesamum e.g. the speciesSesamum indicum [sesame]; Piperaceae such as the genera Piper, Artanthe,Peperomia, Steffensia e.g. the species Piper aduncum, Piper amalago,Piper angustifolium, Piper auritum, Piper betel, Piper cubeba, Piperlongum, Piper nigrum, Piper retrofractum, Artanthe adunca, Artantheelongata, Peperomia elongata, Piper elongatum, Steffensia elongata.[Cayenne pepper, wild pepper]; Poaceae such as the genera Hordeum,Secale, Avena, Sorghum, Andropogon, Holcus, Panicum, Oryza, Zea,Triticum e.g. the species Hordeum vulgare, Hordeum jubatum, Hordeummurinum, Hordeum secalinum, Hordeum distichon Hordeum aegiceras, Hordeumhexastichon, Hordeum hexastichum, Hordeum irregulare, Hordeum satfivum,Hordeum secalinum [barley, pearl barley, foxtail barley, wall barley,meadow barley], Secale cereale [rye], Avena sativa, Avena fatua, Avenabyzantina, Avena fatua var. sativa, Avena hybrida [oat], Sorghumbicolor, Sorghum halepense, Sorghum saccharatum, Sorghum vulgare,Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghumaethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum,Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense,Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghumsubglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcushalepensis, Sorghum miliaceum millet, Panicum militaceum [Sorghum,millet], Oryza sativa, Oryza latifolia [rice], Zea mays [corn,maize]Triticum aestivum, Triticum durum, Triticum turgidum, Triticumhybernum, Triticum macha, Triticum sativum or Triticum vulgare [wheat,bread wheat, common wheat], Proteaceae such as the genera Macadamia e.g.the species Macadamia intergrifolia [macadamia]; Rubiaceae such as thegenera Coffea e.g. the species Cofea spp., Coffea arabica, Coffeacanephora or Coffea liberica [coffee]; Scrophulariaceae such as thegenera Verbascum e.g. the species Verbascum blattaria, Verbascumchaixii, Verbascum densiflorum, Verbascum lagurus, Verbascumlongifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum,Verbascum phlomoides, Verbascum phoenicum, Verbascum pulverulentum orVerbascum thapsus [mullein, white moth mullein, nettle-leaved mullein,dense-flowered mullein, silver mullein, long-leaved mullein, whitemullein, dark mullein, greek mullein, orange mullein, purple mullein,hoary mullein, great mullein]; Solanaceae such as the genera Capsicum,Nicotiana, Solanum, Lycopersicon e.g. the species Capsicum annuum,Capsicum annuum var. glabriusculum, Capsicum frutescens [pepper],Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata, Nicotianaattenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotianaobtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotianarustica, Nicotiana sylvestris [tobacco], Solanum tuberosum [potato],Solanum melongena [egg-plant] (Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme, Solanum integrifolium or Solanumlycopersicum [tomato]; Sterculiaceae such as the genera Theobroma e.g.the species Theobroma cacao [cacao]; Theaceae such as the generaCamellia e.g. the species Camellia sinensis) [tea].

The introduction of the nucleic acids according to the invention, theexpression cassette or the vector into organisms, plants for example,can in principle be done by all of the methods known to those skilled inthe art. The introduction of the nucleic acid sequences gives rise torecombinant or transgenic organisms.

In the case of microorganisms, those skilled in the art can findappropriate methods in the textbooks by Sambrook, J. et al. (1989)Molecular cloning: A laboratory manual, Cold Spring Harbor LaboratoryPress, by F. M. Ausubel et al. (1994) Current protocols in molecularbiology, John Wiley and Sons, by D. M. Glover et al., DNA Cloning Vol.1, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994)Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press orGuthrie et al. Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, 1994, Academic Press.

The transfer of foreign genes into the genome of a plant is calledtransformation. In doing this the methods described for thetransformation and regeneration of plants from plant tissues or plantcells are utilized for transient or stable transformation. Suitablemethods are protoplast transformation by poly(ethylene glycol)-inducedDNA uptake, the “biolistic” method using the gene cannon—referred to asthe particle bombardment method, electroporation, the incubation of dryembryos in DNA solution, microinjection and gene transfer mediated byAgrobacterium. Said methods are described by way of example in B. Jeneset al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press(1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec.Biol. 42 (1991) 205-225). The nucleic acids or the construct to beexpressed is preferably cloned into a vector which is suitable fortransforming Agrobacterium tumefaciens, for example pBin19 (Bevan etal., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by sucha vector can then be used in known manner for the transformation ofplants, in particular of crop plants such as by way of example tobaccoplants, for example by bathing bruised leaves or chopped leaves in anagrobacterial solution and then culturing them in suitable media. Thetransformation of plants by means of Agrobacterium tumefaciens isdescribed, for example, by Hofgen and Willmitzer in Nucl. Acid Res.(1988) 16, 9877 or is known inter alia from F. F. White, Vectors forGene Transfer in Higher Plants; in Transgenic Plants, Vol. 1,Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press,1993, pp. 15-38.

Agrobacteria transformed by an expression vector according to theinvention may likewise be used in known manner for the transformation ofplants such as test plants like Arabidopsis or crop plants such ascereal crops, corn, oats, rye, barley, wheat, soybean, rice, cotton,sugar beet, canola, sunflower, flax, hemp, potatoes, tobacco, tomatoes,carrots, paprika, oilseed rape, tapioca, cassava, arrowroot, tagetes,alfalfa, lettuce and the various tree, nut and vine species, inparticular of oil-containing crop plants such as soybean, peanut, castoroil plant, sunflower, corn, cotton, flax, oilseed rape, coconut, oilpalm, safflower (Carthamus tinctorius) or cocoa bean, e.g. by bathingbruised leaves or chopped leaves in an agrobacterial solution and thenculturing them in suitable media.

The genetically modified plant cells may be regenerated by all of themethods known to those skilled in the art. Appropriate methods can befound in the publications referred to above by S. D. Kung and R. Wu,Potrykus or Höfgen and Willmitzer.

Accordingly, a further aspect of the invention relates to transgenicorganisms transformed by at least one nucleic acid sequence, expressioncassette or vector according to the invention as well as cells, cellcultures, tissue, parts—such as, for example, leaves, roots, etc. in thecase of plant organisms—or reproductive material derived from suchorganisms. The terms “host organism”, “host cell”, “recombinant (host)organism” and “transgenic (host) cell” are used here interchangeably. Ofcourse these terms relate not only to the particular host organism orthe particular target cell but also to the descendants or potentialdescendants of these organisms or cells. Since, due to mutation orenvironmental effects certain modifications may arise in successivegenerations, these descendants need not necessarily be identical withthe parental cell but nevertheless are still encompassed by the term asused here.

For the purposes of the invention “transgenic” or “recombinant” meanswith regard for example to a nucleic acid sequence, an expressioncassette (=gene construct, nucleic acid construct) or a vectorcontaining the nucleic acid sequence according to the invention or anorganism transformed by the nucleic acid sequences, expression cassetteor vector according to the invention all those constructions produced bygenetic engineering methods in which either

-   -   a) the nucleic acid sequence depicted in FIGS. 1 a, 1 b or 1 c        or its derivatives or parts thereof or    -   b) a genetic control sequence functionally linked to the nucleic        acid sequence described under (a), for example a 3′- and/or        5′-genetic control sequence such as a promoter or terminator, or    -   c) (a) and (b)        are not found in their natural, genetic environment or have been        modified by genetic engineering methods, wherein the        modification may by way of example be a substitution, addition,        deletion, inversion or insertion of one or more nucleotide        residues. Natural genetic environment means the natural genomic        or chromosomal locus in the organism of origin or inside the        host organism or presence in a genomic library. In the case of a        genomic library the natural genetic environment of the nucleic        acid sequence is preferably retained at least in part. The        environment borders the nucleic acid sequence at least on one        side and has a sequence length of at least 50 bp, preferably at        least 500 bp, particularly preferably at least 1,000 bp, most        particularly preferably at least 5,000 bp. A naturally occurring        expression cassette—for example the naturally occurring        combination of the natural promoter of the nucleic acid sequence        according to the invention with the corresponding        Δ-8-desaturase, Δ-9-elongase and/or Δ-5-desaturase gene—turns        into a transgenic expression cassette when the latter is        modified by unnatural, synthetic (“artificial”) methods such as        by way of example a mutagenation. Appropriate methods are        described by way of example in U.S. Pat. No. 5,565,350 or WO        00/15815.

Suitable organisms or host organisms for the nucleic acid, expressioncassette or vector according to the invention are advantageously inprinciple all organisms, which are suitable for the expression ofrecombinant genes as described above. Further examples which may bementioned are plants such as Arabidopsis, Asteraceae such as Calendulaor crop plants such as soybean, peanut, castor oil plant, sunflower,flax, corn, cotton, flax, oilseed rape, coconut, oil palm, safflower(Carthamus tinctorius) or cocoa bean.

A further object of the invention relates to the use of a nucleic acidconstruct, e.g. an expression cassette, containing DNA sequencesencoding polypeptides of FIGS. 1 a, 1 b or 1 c or DNA sequenceshybridizing therewith for the transformation of plant cells, tissues orparts of plants.

In doing so, depending on the choice of promoter, the sequences FIGS. 1a, 1 b or 1 c can be expressed specifically in the leaves, in the seeds,the nodules, in roots, in the stem or other parts of the plant. Thosetransgenic plants overproducing sequences of FIGS. 1 a, 1 b or 1 c, thereproductive material thereof, together with the plant cells, tissues orparts thereof are a further object of the present invention.

The expression cassette or the nucleic acid sequences or constructaccording to the invention containing sequences of FIGS. 1 a, 1 b or 1 ccan, moreover, also be employed for the transformation of the organismsidentified by way of example above such as bacteria, yeasts, filamentousfungi and plants.

Within the framework of the present invention, altering metabolicactivity means, for example, the artificially acquired trait ofincreased biosynthetic performance due to functional over expression ofsequences of FIGS. 1 a, 1 b or 1 c in the organisms according to theinvention, advantageously in the transgenic plants according to theinvention, by comparison with the nongenetically modified initial plantsat least for the duration of at least one plant generation.

A constitutive expression of the exogenous sequences of the FIGS. 1 a, 1b or 1 c is, moreover, advantageous. On the other hand, however, aninducible expression may also appear desirable.

The efficiency of the expression of the sequences of the FIGS. 1 a, 1 bor 1 c can be determined, for example, in vitro by shoot meristempropagation. In addition, an expression of the sequences of FIGS. 1 a, 1b or 1 c modified in nature and level and its effect on the metabolicpathways performance can be tested on test plants in greenhouse trials.

An additional object of the invention comprises transgenic organismssuch as transgenic plants transformed by an expression cassettecontaining sequences of FIGS. 1 a, 1 b or 1 c according to the inventionor DNA sequences hybridizing therewith, as well as transgenic cells,tissue, parts and reproduction material of such plants. Particularpreference is given in this case to transgenic crop plants such as byway of example barley, wheat, rye, oats, corn, soybean, rice, cotton,sugar beet, oilseed rape and canola, sunflower, flax, hemp, thistle,potatoes, tobacco, tomatoes, tapioca, cassava, arrowroot, alfalfa,lettuce and the various tree, nut and vine species.

For the purposes of the invention plants are mono- and dicotyledonousplants, mosses or algae.

A further refinement according to the invention are transgenic plants asdescribed above which contain a nucleic acid sequence or constructaccording to the invention or a expression cassette according to theinvention. Furthermore, by derivatives is meant homologues of thesequences of FIGS. 1 a, 1 b or 1 c, for example eukaryotic homologues,truncated sequences, single-stranded DNA of the encoding and nonencodingDNA sequence or RNA of the encoding and nonencoding DNA sequence.

In addition, by homologues of the sequences of FIGS. 1 a, 1 b or 1 c ismeant derivatives such as by way of example promoter variants. Thesevariants may be modified by one or more nucleotide exchanges, byinsertion(s) and/or deletion(s) without, however, adversely affectingthe functionality or efficiency of the promoters.

Furthermore, the promoters can have their efficiency increased byaltering their sequence or be completely replaced by more effectivepromoters even of foreign organisms.

By derivatives is also advantageously meant variants whose nucleotidesequence has been altered in the region from −1 to −2000 ahead of thestart codon in such a way that the gene expression and/or the proteinexpression is modified, preferably increased. Furthermore, byderivatives is also meant variants which have been modified at the 3′end.

Suitable promoters in the expression cassette are in principle allpromoters which can control the expression of foreign genes in organismssuch as microorganisms like protozoa such as ciliates, algae such asgreen, brown, red or blue algae such as Euglenia, bacteria such asgram-positive or gram-negative bacteria, yeasts such as Saccharomyces,Pichia or Schizosaccharomyces or fungi such as Mortierella,Thraustochytrium or Schizochytrium or plants, advantageously in plantsor fungi. Use is preferably made in particular of plant promoters orpromoters derived from a plant virus. Advantageous regulation sequencesfor the method according to the invention are found for example inpromoters such as cos, tac, trp, tet, trp-tet, Ipp, lac, Ipp-lac,lacIq-, T7, T5, T3, gal, trc, ara, SP6, λ-PR or in λ-PL promoters whichare employed advantageously in gram-negative bacteria. Otheradvantageous regulation sequences are found, for example, in thegram-positive promoters amy and SPO2, in the yeast or fungal promotersADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH or in the plantpromoters CaMV/35S [Franck et al., Cell 21 (1980) 285-294], SSU, OCS,lib4, STLS1, B33, nos (=Nopalin Synthase Promoter) or in the ubiquintinor phaseolin promoter. The expression cassette may also contain achemically inducible promoter by means of which the expression of theexogenous sequences of the odd numbers of SEQ. ID No. 1-269 in theorganisms can be controlled advantageously in the plants at a particulartime. Advantageous plant promoters of this type are by way of examplethe PRP1 promoter [Ward et al., Plant. Mol. Biol. 22 (1993), 361-366], apromoter inducible by benzenesulfonamide (EP 388 186), a promoterinducible by tetracycline [Gatz et al., (1992) Plant J. 2, 397-404], apromoter inducible by salicylic acid (WO 95/19443), a promoter inducibleby abscisic acid (EP 335 528) and a promoter inducible by ethanol orcyclohexanone (WO93/21334). Other examples of plant promoters which canadvantageously be used are the promoter of cytosolic FBPase from potato,the ST-LSI promoter from potato (Stockhaus et al., EMBO J. 8 (1989)2445-245), the promoter of phosphoribosyl pyrophosphate amidotransferasefrom Glycine max (see also gene bank accession number U87999) or anodiene-specific promoter as described in EP 249 676. Particularlyadvantageous are those plant promoters which ensure expression intissues or plant parts/organs in which fatty acid biosynthesis or theprecursor stages thereof occurs, as in endosperm or in the developingembryo for example. Particularly noteworthy are advantageous promoterswhich ensure seed-specific expression such as by way of example the USPpromoter or derivatives thereof, the LEB4 promoter, the phaseolinpromoter or the napin promoter. The particularly advantageous USPpromoter cited according to the invention or its derivatives mediatevery early gene expression in seed development [Baeumlein et al., MolGen Genet, 1991, 225 (3): 459-67]. Other advantageous seed-specificpromoters which may be used for monocotylodonous or dicotylodonousplants are the promoters suitable for dicotylodons such as napin genepromoters, likewise cited by way of example, from oilseed rape (U.S.Pat. No. 5,608,152), the oleosin promoter from Arabidopsis (WO98/45461), the phaseolin promoter from Phaseolus vulgaris (U.S. Pat. No.5,504,200), the Bce4 promoter from Brassica (WO 91/13980) or theleguminous B4 promoter (LeB4, Baeumlein et al., Plant J., 2, 2, 1992:233-239) or promoters suitable for monocotylodons such as the promotersof the Ipt2 or Ipt1 gene in barley (WO 95/15389 and WO 95/23230) or thepromoters of the barley hordeine gene, the rice glutelin gene, the riceoryzin gene, the rice prolamin gene, the wheat gliadin gene, the whiteglutelin gene, the corn zein gene, the oats glutelin gene, the sorghumkasirin gene or the rye secalin gene which are described in WO99/16890.

Furthermore, particularly preferred are those promoters, which ensurethe expression in tissues, or plant parts in which, for example, thebiosynthesis of fatty acids, oils and lipids or the precursor stagesthereof takes place. Particularly noteworthy are promoters, which ensurea seed-specific expression. Noteworthy are the promoter of the napingene from oilseed rape (U.S. Pat. No. 5,608,152), the USP promoter fromVicia faba (USP=unknown seed protein, Baeumlein et al., Mol Gen Genet,1991, 225 (3): 459-67), the promoter of the oleosin gene fromArabidopsis (WO98/45461), the phaseolin promoter (U.S. Pat. No.5,504,200) or the promoter of the legumin B4 gene (LeB4; Baeumlein etal., 1992, Plant Journal, 2 (2): 233-9). Other promoters to be mentionedare that of the Ipt2 or Ipt1 gene from barley (WO95/15389 andWO95/23230) which mediate seed-specific expression in monocotyledonousplants. Other advantageous seed specific promoters are promoters such asthe promoters from rice, corn or wheat disclosed in WO 99/16890 orAmy32b, Amy6-6 or aleurain (U.S. Pat. No. 5,677,474), Bce4 (rape, U.S.Pat. No. 5,530,149), glycinin (soy bean, EP 571 741), phosphoenolpyruvat carboxylase (soy bean, JP 06/62870), ADR12-2 (soy bean, WO98/08962), isocitratlyase (rape, U.S. Pat. No. 5,689,040) or β-amylase(barley, EP 781 849).

As described above, the expression construct (=gene construct, nucleicacid construct) may contain yet other genes, which are to be introducedinto the organisms. These genes can be subject to separate regulation orbe subject to the same regulation region as sequences FIGS. 1 a, 1 b or1 c. These genes are by way of example other biosynthesis genes,advantageously for fatty acid biosynthesis, vitamin biosynthesis etc.that allow increased synthesis.

In principle all natural promoters with their regulation sequences canbe used like those named above for the expression cassette according tothe invention and the method according to the invention. Over and abovethis, synthetic promoters may also advantageously be used.

In the preparation of an expression cassette various DNA fragments canbe manipulated in order to obtain a nucleotide sequence, which usefullyreads in the correct direction and is equipped with a correct readingraster. To connect the DNA fragments (=nucleic acids according to theinvention) to one another adaptors or linkers may be attached to thefragments.

The promoter and the terminator regions can usefully be provided in thetranscription direction with a linker or polylinker containing one ormore restriction points for the insertion of this sequence. Generally,the linker has 1 to 10, mostly 1 to 8, preferably 2 to 6, restrictionpoints. In general the size of the linker inside the regulatory regionis less than 100 bp, frequently less than 60 bp, but at least 5 bp. Thepromoter may be both native or homologous as well as foreign orheterologous to the host organism, for example to the host plant. In the5′-3′ transcription direction the expression cassette contains thepromoter, a DNA sequence which encodes of FIGS. 1 a, 1 b or 1 c gene anda region for transcription termination. Different termination regionscan be exchanged for one another in any desired fashion.

Furthermore, manipulations which provide suitable restriction interfacesor which remove excess DNA or restriction interfaces can be employed.Where insertions, deletions or substitutions, such as transitions andtransversions, come into consideration, in vitro mutagenesis, primerrepair, restriction or ligation may be used. In suitable manipulationssuch as restriction, chewing back or filling of overhangs for blunt endscomplementary ends of the fragments can be provided for the ligation.

For an advantageous high expression the attachment of the specific ERretention signal SEKDEL inter alia can be of importance (Schouten, A. etal., Plant Mol. Biol. 30 (1996), 781-792). In this way the averageexpression level is tripled or even quadrupled. Other retention signalswhich occur naturally in plant and animal proteins located in the ER mayalso be employed for the construction of the cassette. In anotherpreferred embodiment a plastidial targeting sequence is used asdescribed by Napier J. A. [Targeting of foreign proteins to thechloroplast, Methods Mol. Biol., 49, 1995: 369-376]. A preferred usedvector comprising said plastidial targeting sequence is disclosed byColin Lazarus [Guerineau F., Woolston S., Brooks L., Mullineaux P. “Anexpression cassette for targeting foreign proteins into chloroplast;Nucleic. Acids Res., Dec. 9, 16 (23), 1988: 11380].

Preferred polyadenylation signals are plant polyadenylation signals,preferably those which substantially correspond to T-DNA polyadenylationsignals from Agrobacterium tumefaciens, in particular gene 3 of theT-DNA (octopin synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBOJ. 3 (1984), 835 et seq.) or corresponding functional equivalents.

An expression cassette is produced by fusion of a suitable promoter withsuitable sequences of FIGS. 1 a, 1 b or 1 c together with apolyadenylation signal by common recombination and cloning techniques asdescribed, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1989) as well as in T. J. Silhavy, M. L.Berman and L. W. Enquist, Experiments with Gene Fusions, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M.et al., Current Protocols in Molecular Biology, Greene Publishing Assoc.and Wiley-Interscience (1987).

In the preparation of an expression cassette various DNA fragments canbe manipulated to produce a nucleotide sequence which usefully reads inthe correct direction and is equipped with a correct reading raster.Adapters or linkers can be attached to the fragments for joining the DNAfragments.

The promoter and the terminator regions can usefully be provided in thetranscription direction with a linker or polylinker containing one ormore restriction points for the insertion of this sequence. Generally,the linker has 1 to 10, mostly 1 to 8, preferably 2 to 6, restrictionpoints. In general the size of the linker inside the regulatory regionis less than 100 bp, frequently less than 60 bp, but at least 5 bp. Thepromoter may be both native or homologous as well as foreign orheterologous to the host organism, for example to the host plant. In the5′-3′ transcription direction the expression cassette contains thepromoter, a DNA sequence which either encodes gene of the odd numbers ofSEQ. ID No. 1-269 and a region for transcription termination. Differenttermination regions can be exchanged for one another in any desiredfashion.

In the preparation of an expression cassette various DNA fragments canbe manipulated to produce a nucleotide sequence which usefully reads inthe correct direction and is equipped with a correct reading raster.Adapters or linkers can be attached to the fragments for joining the DNAfragments.

The DNA sequences encoding the nucleic acid sequences used in theinventive processes such as the sequences of the FIGS. 1 a, 1 b or 1 ccontain all the sequence characteristics needed to achieve correctlocalization of respective biosynthesis. Accordingly, no furthertargeting sequences are needed per se. However, such a localization maybe desirable and advantageous and hence artificially modified orreinforced so that such fusion constructs are also a preferredadvantageous embodiment of the invention.

Particularly preferred are sequences which ensure targeting in plastids.Under certain circumstances targeting into other compartments (reportedin: Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423) may also bedesirable, e.g. into vacuoles, the mitochondrium, the endoplasmicreticulum (ER), peroxisomes, lipid structures or due to lack ofcorresponding operative sequences retention in the compartment oforigin, the cytosol.

As used herein, the term “environmental stress” refers to anysub-optimal growing condition and includes, but is not limited to,sub-optimal conditions associated with salinity, drought, temperature,metal, chemical, pathogenic and oxidative stresses, or combinationsthereof. In preferred embodiments, the environmental stress can besalinity, drought, heat, or low temperature, or combinations thereof,and in particular, can be low water content or low temperature. Whereindrought stress means any environmental stress which leads to a lack ofwater in plants or reduction of water supply to plants, wherein lowtemperature stress means freezing of plants below +4° C. as well aschilling of plants below 15° C. and wherein high temperature stressmeans for example a temperature above 35° C. The range of stress andstress response depends on the different plants which are used for theinvention, i.e. it differs for example between a plant such as wheat anda plant such as Arabidopsis. A common response of plants toenvironmental stress is the loss of yield or the loss of quality. It isalso to be understood that as used in the specification and in theclaims, “a” or “an” can mean one or more, depending upon the context inwhich it is used. Thus, for example, reference to “a cell” can mean thatat least one cell can be utilized.

As also used herein, the terms “nucleic acid” and “nucleic acidmolecule” are intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. This term also encompassesuntranslated sequence located at both the 3′ and 5′ ends of the codingregion of the gene: at least about 1000 nucleotides of sequence upstreamfrom the 5′ end of the coding region and at least about 200 nucleotidesof sequence downstream from the 3′ end of the coding region of the gene.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one that is substantiallyseparated from other nucleic acid molecules, which are present in thenatural source of the nucleic acid. That means other nucleic acidmolecules are present in an amount less than 5% based on weight of theamount of the desired nucleic acid, preferably less than 2% by weight,more preferably less than 1% by weight, most preferably less than 0.5%by weight. Preferably, an “isolated” nucleic acid is free of some of thesequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. For example, in variousembodiments, the isolated stress related protein encoding nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of nucleotide sequences which naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be free from some of the other cellular material withwhich it is naturally associated, or culture medium when produced byrecombinant techniques, or chemical precursors or other chemicals whenchemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule encoding an SRP or a portion thereof which confers toleranceand/or resistance to environmental stress in plants, can be isolatedusing standard molecular biological techniques and the sequenceinformation provided herein. For example, a Arabidopsis thaliana stressrelated protein encoding cDNA can be isolated from a A. thaliana c-DNAlibrary using all or portion of one of the sequences of FIGS. 1 a, 1 bor 1 c. Moreover, a nucleic acid molecule encompassing all or a portionof one of the sequences of FIGS. 1 a, 1 b or 1 c can be isolated by thepolymerase chain reaction using oligonucleotide primers designed basedupon this sequence. For example, mRNA can be isolated from plant cells(e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwinet al., 1979 Biochemistry 18:5294-5299) and cDNA can be prepared usingreverse transcriptase (e.g., Moloney MLV reverse transcriptase,available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase,available from Seikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for polymerase chain reaction amplification canbe designed based upon one of the nucleotide sequences shown in FIGS. 1a, 1 b or 1 c. A nucleic acid molecule of the invention can be amplifiedusing cDNA or, alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid molecule so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a SRP encoding nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises one of the nucleotide sequences shown in sequencesFIGS. 1 a, 1 b or 1 c encoding the SRP (i.e., the “coding region”), aswell as 5′ untranslated sequences and 3′ untranslated sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of one of the sequences δf the nucleic acidof FIGS. 1 a, 1 b or 1 c, for example, a fragment which can be used as aprobe or primer or a fragment encoding a biologically active portion ofa SRP.

Portions of proteins encoded by the SRP encoding nucleic acid moleculesof the invention are preferably biologically active portions describedherein. As used herein, the term “biologically active portion of” a SRPis intended to include a portion, e.g., a domain/motif, of stressrelated protein that participates in a stress tolerance and/orresistance response in a plant. To determine whether a SRP, or abiologically active portion thereof, results in increased stresstolerance in a plant, a stress analysis of a plant comprising the SRPmay be performed. Such analysis methods are well known to those skilledin the art, as detailed in the Examples. More specifically, nucleic acidfragments encoding biologically active portions of a SRP can be preparedby isolating a portion of one of the sequences of the nucleic acid ofFIGS. 1 a, 1 b or 1 c expressing the encoded portion of the SRP orpeptide (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of the SRP or peptide.

Biologically active portions of a SRP are encompassed by the presentinvention and include peptides comprising amino acid sequences derivedfrom the amino acid sequence of a SRP encoding gene, or the amino acidsequence of a protein homologous to a SRP, which include fewer aminoacids than a full length SRP or the full length protein which ishomologous to a SRP, and exhibits at least some enzymatic activity of aSRP. Typically, biologically active portions (e.g., peptides which are,for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or moreamino acids in length) comprise a domain or motif with at least oneactivity of a SRP. Moreover, other biologically active portions in whichother regions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the activities describedherein. Preferably, the biologically active portions of a SRP includeone or more selected domains/motifs or portions thereof havingbiological activity.

The term “biological active portion” or “biological activity” means aSRP or a portion of a SRP which still has at least 10% or 20%,preferably 20%, 30%, 40% or 50%, especially preferably 60%, 70% or 80%of the enzymatic activity of the natural or starting enzyme.

A nucleic acid molecule encompassing a complete sequence of the nucleicacid molecules used in the process, for example the polynucleotide ofthe invention, or a part thereof may additionally be isolated bypolymerase chain reaction, oligonucleotide primers based on thissequence or on parts thereof being used. For example, a nucleic acidmolecule comprising the complete sequence or part thereof can beisolated by polymerase chain reaction using oligonucleotide primerswhich have been generated on the basis of this sequence—For example,mRNA can be isolated from cells (for example by means of the guanidiniumthiocyanate extraction method of Chirgwin et al. (1979) Biochemistry18:5294-5299) and cDNA can be generated by means of reversetranscriptase (for example Moloney MLV reverse transcriptase, availablefrom Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase, obtainablefrom Seikagaku America, Inc., St. Petersburg, Fla.).

Synthetic oligonucleotide primers for the amplification, e.g. as shownin table 2, by means of polymerase chain reaction can be generated onthe basis of a sequence shown herein, for example the sequence shown inFIGS. 1 a, 1 b or 1 c or the sequences derived from polypeptides asshown in FIGS. 1 a, 1 b or 1 c.

Moreover, it is possible to identify conserved regions from variousorganisms by carrying out protein sequence alignments with thepolypeptide used in the process of the invention, in particular withsequences of the polypeptide of the invention, from which conservedregions, and in turn, degenerate primers can be derived. Conservedregion for the polypeptide of the invention are indicated in thealignment shown in the drawing. Conserved regions are those, which showa very little variation in the amino acid in one particular position ofseveral homologs from different origin. The consenus sequences shown inFIG. 2 are derived from said alignments.

Degenerated primers can then be utilized by PCR for the amplification offragments of novel proteins having above-mentioned activity, e.g. havingan SPR activity or further functional homologs of the polypeptide of theinvention from other organisms.

These fragments can then be utilized as hybridization probe forisolating the complete gene sequence. As an alternative, the missing 5′and 3′ sequences can be isolated by means of RACE-PCR (rapidamplification of cDNA ends). A nucleic acid molecule according to theinvention can be amplified using cDNA or, as an alternative, genomic DNAas template and suitable oligonucleotide primers, following standard PCRamplification techniques. The nucleic acid molecule amplified thus canbe cloned into a suitable vector and characterized by means of DNAsequence analysis.

Oligonucleotides, which correspond to one of the nucleic acid moleculesused in the process can be generated by standard synthesis methods, forexample using an automatic DNA synthesizer.

Nucleic acid molecules which are advantageously for the processaccording to the invention can be isolated based on their homology tothe nucleic acid molecules disclosed herein using the sequences or partthereof as hybridization probe and following standard hybridizationtechniques under stringent hybridization conditions. In this context, itis possible to use, for example, isolated nucleic acid molecules of atleast 15, 20, 25, 30, 35, 40, 50, 60 or more nucleotides, preferably ofat least 15, 20 or 25 nucleotides in length which hybridize understringent conditions with the above-described nucleic acid molecules, inparticular with those which encompass a nucleotide sequence of thenucleic acid molecule used in the process of the invention or encoding aprotein used in the invention or of the nucleic acid molecule of theinvention. Nucleic acid molecules with 30, 50, 100, 250 or morenucleotides may also be used.

In addition to fragments of the SRP described herein, the presentinvention includes homologs and analogs of naturally occurring SRP andSRP encoding nucleic acids in a plant.

“Homologs” are defined herein as two nucleic acids or proteins that havesimilar, or “homologous”, nucleotide or amino acid sequences,respectively. Homologs include allelic variants, orthologs, paralogs,agonists and antagonists of SRP as defined hereafter. The term “homolog”further encompasses nucleic acid molecules that differ from one of thenucleotide sequences as the polynucleotide as shown in FIGS. 1 a, 1 b or1 c (and portions thereof) due to degeneracy of the genetic code andthus encode the same SRP as that encoded by the amino acid sequences asthe polypeptide as shown in FIGS. 1 a, 1 b or 1 c. As used herein a“naturally occurring” SRP refers to a SRP amino acid sequence thatoccurs in nature.

The term “homology” means that the respective nucleic acid molecules orencoded proteins are functionally and/or structurally equivalent. Thenucleic acid molecules that are homologous to the nucleic acid moleculesdescribed above and that are derivatives of said nucleic acid moleculesare, for example, variations of said nucleic acid molecules whichrepresent modifications having the same biological function, inparticular encoding proteins with the same or substantially the samebiological function. They may be naturally occurring variations, such assequences from other plant varieties or species, or mutations. Thesemutations may occur naturally or may be obtained by mutagenesistechniques. The allelic variations may be naturally occurring allelicvariants as well as synthetically produced or genetically engineeredvariants. Structurally equivalents can, for example, be identified bytesting the binding of said polypeptide to antibodies or computer basedpredictions. Structurally equivalent have the similar immunologicalcharacteristic, e.g. comprise similar epitopes.

Functional equivalents derived from one of the polypeptides as shown inany sequence according to the invention by substitution, insertion ordeletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least55%, 60%, 65% or 70% by preference at least 80%, especially preferablyat least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably atleast 95%, 97%, 98% or 99% homology with one of the polypeptides asshown in FIGS. 1 a, 1 b or 1 c according to the invention and aredistinguished by essentially the same properties as the polypeptide asshown in FIGS. 1 a, 1 b or 1 c.

Functional equivalents derived from the nucleic acid sequence as shownin any sequence according to the invention by substitution, insertion ordeletion have at least 30%, 35%, 40%, 45% or 50%, preferably at least55%, 60%, 65% or 70% by preference at least 80%, especially preferablyat least 85% or 90%, 91%, 92%, 93% or 94%, very especially preferably atleast 95%, 97%, 98% or 99% homology with one of the polypeptides asshown in FIGS. 1 a, 1 b or 1 c according to the invention and encodepolypeptides having essentially the same properties as the polypeptideas shown in FIGS. 1 a, 1 b or 1 c.

“Essentially the same properties” of a functional equivalent is aboveall understood as meaning that the functional equivalent has abovementioned activity, e.g conferring an increase in the fine chemicalamount while increasing the amount of protein, activity or function ofsaid functional equivalent in an organism, e.g. a microorganism, a plantor plant or animal tissue, plant or animal cells or a part of the same.

By “hybridizing” it is meant that such nucleic acid molecules hybridizeunder conventional hybridization conditions, preferably under stringentconditions such as described by, e.g., Sambrook (Molecular Cloning; ALaboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989)) or in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

According to the invention, DNA as well as RNA molecules of the nucleicacid of the invention can be used as probes. Further, as template forthe identification of functional homologues Northern blot assays as wellas Southern blot assays can be performed. The Northern blot assayadvantageously provides further informations about the expressed geneproduct: e.g. expression pattern, occurrence of processing steps, likesplicing and capping, etc. The Southern blot assay provides additionalinformation about the chromosomal localization and organization of thegene encoding the nucleic acid molecule of the invention.

A preferred, nonlimiting example of stringent hydridization conditionsare hybridizations in 6× sodium chloride/sodium citrate (=SSC) atapproximately 45° C., followed by one or more wash steps in 0.2×SSC,0.1% SDS at 50 to 65° C., for example at 50° C., 55° C. or 60° C. Theskilled worker knows that these hybridization conditions differ as afunction of the type of the nucleic acid and, for example when organicsolvents are present, with regard to the temperature and concentrationof the buffer. The temperature under “standard hybridization conditions”differs for example as a function of the type of the nucleic acidbetween 42° C. and 58° C., preferably between 45° C. and 50° C. in anaqueous buffer with a concentration of 0.1×0.5 x, 1×, 2×, 3×, 4× or5×SSC (pH 7.2). If organic solvent(s) is/are present in theabovementioned buffer, for example 50% formamide, the temperature understandard conditions is approximately 40° C., 42° C. or 45° C. Thehybridization conditions for DNA:DNA hybrids are preferably for example0.1×SSC and 20° C., 25° C., 30° C., 35° C., 40° C. or 45° C., preferablybetween 30° C. and 45° C. The hybridization conditions for DNA:RNAhybrids are preferably for example 0.1×SSC and 30° C., 35° C., 40° C.,45° C., 50° C. or 55° C., preferably between 45° C. and 55° C. Theabovementioned hybridization temperatures are determined for example fora nucleic acid approximately 100 bp (=base pairs) in length and a G+Ccontent of 50% in the absence of formamide. The skilled worker knows todetermine the hybridization conditions required with the aid oftextbooks, for example the ones mentioned above, or from the followingtextbooks: Sambrook et al., “Molecular Cloning”, Cold Spring HarborLaboratory, 1989; Hames and Higgins (Ed.) 1985, “Nucleic AcidsHybridization: A Practical Approach”, IRL Press at Oxford UniversityPress, Oxford; Brown (Ed.) 1991, “Essential Molecular Biology: APractical Approach”, IRL Press at Oxford University Press, Oxford.

A further example of one such stringent hybridization condition ishybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at65° C. for one hour. Alternatively, an exemplary stringent hybridizationcondition is in 50% formamide, 4×SSC at 42° C. Further, the conditionsduring the wash step can be selected from the range of conditionsdelimited by low-stringency conditions (approximately 2×SSC at 50° C.)and high-stringency conditions (approximately 0.2×SSC at 50° C.,preferably at 65° C.) (20×SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). Inaddition, the temperature during the wash step can be raised fromlow-stringency conditions at room temperature, approximately 22° C., tohigher-stringency conditions at approximately 65° C. Both of theparameters salt concentration and temperature can be variedsimultaneously, or else one of the two parameters can be kept constantwhile only the other is varied. Denaturants, for example formamide orSDS, may also be employed during the hybridization. In the presence of50% formamide, hybridization is preferably effected at 42° C. Relevantfactors like i) length of treatment, ii) salt conditions, iii) detergentconditions, iv) competitor DNAs, v) temperature and vi) probe selectioncan be combined case by case so that not all possibilities can bementioned herein.

Thus, in a preferred embodiment, Northern blots are prehybridized withRothi-Hybri-Quick buffer (Roth, Karlsruhe) at 68° C. for 2 h.Hybridzation with radioactive labelled probe is done overnight at 68° C.Subsequent washing steps are performed at 68° C. with 1×SSC.

For Southern blot assays the membrane is prehybridized withRothi-Hybri-Quick buffer (Roth, Karlsruhe) at 68° C. for 2 h. Thehybridzation with radioactive labelled probe is conducted over night at68° C. Subsequently the hybridization buffer is discarded and the filtershortly washed using 2×SSC; 0.1% SDS. After discarding the washingbuffer new 2×SSC; 0.1% SDS buffer is added and incubated at 68° C. for15 minutes. This washing step is performed twice followed by anadditional washing step using 1×SSC; 0.1% SDS at 68° C. for 10 min.

Some further examples of conditions for DNA hybridization (Southern blotassays) and wash step are shown hereinbelow:

-   -   (1) Hybridization conditions can be selected, for example, from        the following conditions:        -   a) 4×SSC at 65° C.,        -   b) 6×SSC at 45° C.,        -   c) 6×SSC, 100 mg/ml denatured fragmented fish sperm DNA at            68° C.,        -   d) 6×SSC, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA at            68° C.,        -   e) 6×SSC, 0.5% SDS, 100 mg/ml denatured fragmented salmon            sperm DNA, 50% formamide at 42° C.,        -   f) 50% formamide, 4×SSC at 42° C.,        -   g) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1%            Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate            buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at 42° C.,        -   h) 2× or 4×SSC at 50° C. (low-stringency condition), or        -   i) 30 to 40% formamide, 2× or 4×SSC at 42° C.            (low-stringency condition).    -   (2) Wash steps can be selected, for example, from the following        conditions:        -   a) 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.        -   b) 0.1×SSC at 65° C.        -   c) 0.1×SSC, 0.5% SDS at 68° C.        -   d) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C.        -   e) 0.2×SSC, 0.1% SDS at 42° C.        -   f) 2×SSC at 65° C. (low-stringency condition).

In an other embodiment is meant by standard conditions, for example,depending on the nucleic acid in question temperatures between 42° C.and 58° C. in an aqueous buffer solution having a concentration ofbetween 0.1 and 5×SSC (1×SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.2)or additionally in the presence of 50% formamide, such as by way ofexample 42° C. in 5×SSC, 50% formamide. Hybridization conditions forDNA:DNA hybrids are advantageously 0.1×SSC and temperatures betweenapproximately 20° C. and 45° C., preferably between approximately 30° C.and 45° C. For DNA:RNA hybrids the hybridization conditions areadvantageously 0.1×SSC and temperatures between approximately 30° C. and55° C., preferably between approximately 45° C. and 55° C. Thesespecified temperatures for hybridization are melting temperature valuescalculated by way of example for a nucleic acid having a length ofapproximately 100 nucleotides and a G+C content of 50% in the absence offormamide. The experimental conditions for DNA hybridization aredescribed in relevant genetics textbooks such as by way of exampleSambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratory,1989, and may be calculated by formulae known to those skilled in theart, for example as a function of the length of the nucleic acids, thenature of the hybrids or the G+C content. Those skilled in the art maydraw on the following textbooks for further information onhybridization: Ausubel et al. (eds), 1985, Current Protocols inMolecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds),1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press atOxford University Press, Oxford; Brown (ed), 1991, Essential MolecularBiology: A Practical Approach, IRL Press at Oxford University Press,Oxford.

Polypeptides having above-mentioned activity, i.e. conferring thealtered metabolic activity, derived from other organisms, can be encodedby other DNA sequences which hybridize to the sequences shown in FIGS. 1a, 1 b or 1 c under relaxed hybridization conditions and which code onexpression for peptides conferring an altered metabolic activity.

Further, some applications have to be performed at low stringencyhybridisation conditions, without any consequences for the specificityof the hybridisation. For example, a Southern blot analysis of total DNAcould be probed with a nucleic acid molecule of the present inventionand washed at low stringency (55° C. in 2×SSPEO, 1% SDS). Thehybridisation analysis could reveal a simple pattern of only genesencoding polypeptides of the present invention or used in the process ofthe invention, e.g. having herein-mentioned activity of increasing thefine chemical. A further example of such low-stringent hybridizationconditions is 4×SSC at 50° C. or hybridization with 30 to 40% formamideat 42° C. Such molecules comprise those which are fragments, analoguesor derivatives of the polypeptide of the invention or used in theprocess of the invention and differ, for example, by way of amino acidand/or nucleotide deletion(s), insertion(s), substitution (s),addition(s) and/or recombination (s) or any other modification(s) knownin the art either alone or in combination from the above-described aminoacid sequences or their underlying nucleotide sequence(s). However, itis preferred to use high stringency hybridisation conditions.

Hybridization should advantageously be carried out with fragments of atleast 5, 10, 15, 20, 25, 30, 35 or 40 bp, advantageously at least 50,60, 70 or 80 bp, preferably at least 90, 100 or 110 bp. Most preferablyare fragments of at least 15, 20, or 30 bp. Preferably are alsohybridizations with at least 100 bp or 200, very especially preferablyat least 400 bp in length. In an especially preferred embodiment, thehybridization should be carried out with the entire nucleic acidsequence with conditions described above.

The terms “fragment”, “fragment of a sequence” or “part of a sequence”mean a truncated sequence of the original sequence referred to. Thetruncated sequence (nucleic acid or protein sequence) can vary widely inlength; the minimum size being a sequence of sufficient size to providea sequence with at least a comparable function and/or activity of theoriginal sequence referred to or hybridizing with the nucleic acidmolecule of the invention or used in the process of the invention understringent conditions, while the maximum size is not critical. In someapplications, the maximum size usually is not substantially greater thanthat required to provide the desired activity and/or function(s) of theoriginal sequence.

In addition to fragments and fusion polypeptides of the SRPs describedherein, the present invention includes homologs and analogs of naturallyoccurring SRPs and SRP encoding nucleic acids in a plant. “Homologs” aredefined herein as two nucleic acids or polypeptides that have similar,or substantially identical, nucleotide or amino acid sequences,respectively. Homologs include allelic variants, orthologs, paralogs,agonists and antagonists of SRPs as defined hereafter. The term“homolog” further encompasses nucleic acid molecules that differ fromone of the nucleotide sequences shown in FIGS. 1 a, 1 b or 1 c (andportions thereof) due to degeneracy of the genetic code and thus encodethe same SRP as that encoded by the nucleotide sequences shown in FIGS.1 a, 1 b or 1 c. As used herein a “naturally occurring” SRP refers to aSRP amino acid sequence that occurs in nature. Preferably, a naturallyoccurring SRP comprises an amino acid sequence selected from the groupconsisting of polypeptides of FIGS. 1 a, 1 b or 1 c.

An agonist of the SRP can retain substantially the same, or a subset, ofthe biological activities of the SRP. An antagonist of the SRP caninhibit one or more of the activities of the naturally occurring form ofthe SRP. For example, the SRP antagonist can competitively bind to adownstream or upstream member of the cell membrane component metaboliccascade that includes the SRP, or bind to a SRP that mediates transportof compounds across such membranes, thereby preventing translocationfrom taking place.

Nucleic acid molecules corresponding to natural allelic variants andanalogs, orthologs and paralogs of a SRP cDNA can be isolated based ontheir identity to the Saccharomyces cerevisiae, E. coli, Brassica napus,Glycine max, or Oryza sativa SRP nucleic acids described herein usingSRP cDNAs, or a portion thereof, as a hybridization probe according tostandard hybridization techniques under stringent hybridizationconditions. In an alternative embodiment, homologs of the SRP can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the SRP for SRP agonist or antagonist activity.In one embodiment, a variegated library of SRP variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of SRP variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential SRP sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion polypeptides (e.g., for phagedisplay) containing the set of SRP sequences therein. There are avariety of methods that can be used to produce libraries of potentialSRP homologs from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene is then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential SRP sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art. See, e.g., Narang, S.A., 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem.53:323; Itakura et al., 1984, Science 198:1056; Ike et al., 1983,Nucleic Acid Res. 11:477.

In addition, libraries of fragments of the SRP coding regions can beused to generate a variegated population of SRP fragments for screeningand subsequent selection of homologs of a SRP. In one embodiment, alibrary of coding sequence fragments can be generated by treating adouble stranded PCR fragment of a SRP coding sequence with a nucleaseunder conditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA, which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminal,and internal fragments of various sizes of the SRP.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of SRP homologs. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique that enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify SRP homologs (Arkin and Yourvan, 1992, PNAS 89:7811-7815;Delgrave et al., 1993, Polypeptide Engineering 6(3):327-331). In anotherembodiment, cell based assays can be exploited to analyze a variegatedSRP library, using methods well known in the art. The present inventionfurther provides a method of identifying a novel SRP, comprising (a)raising a specific antibody response to a SRP, or a fragment thereof, asdescribed herein; (b) screening putative SRP material with the antibody,wherein specific binding of the antibody to the material indicates thepresence of a potentially novel SRP; and (c) analyzing the boundmaterial in comparison to known SRP, to determine its novelty.

As stated above, the present invention includes SRPs and homologsthereof. To determine the percent sequence identity of two amino acidsequences (e.g., one of the sequences of FIGS. 1 a, 1 b or 1 c, and amutant form thereof), the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of onepolypeptide for optimal alignment with the other polypeptide or nucleicacid). The amino acid residues at corresponding amino acid positions arethen compared. When a position in one sequence (e.g., one of thesequences of FIGS. 1 a, 1 b or 1 c) is occupied by the same amino acidresidue as the corresponding position in the other sequence (e.g., amutant form of the sequence selected from the polypeptide of FIGS. 1 a,1 b or 1 c), then the molecules are identical at that position. The sametype of comparison can be made between two nucleic acid sequences.

The percent sequence identity between the two sequences is a function ofthe number of identical positions shared by the sequences (i.e., percentsequence identity=numbers of identical positions/total numbers ofpositions×100). Preferably, the isolated amino acid homologs included inthe present invention are at least about 50-60%, preferably at leastabout 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%,85-90% or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%or more identical to an entire amino acid sequence shown in FIGS. 1 a, 1b or 1 c. In yet another embodiment, the isolated amino acid homologsincluded in the present invention are at least about 50-60%, preferablyat least about 60-70%, and more preferably at least about 70-75%,75-80%, 80-85%, 85-90% or 90-95%, and most preferably at least about96%, 97%, 98%, 99% or more identical to an entire amino acid sequenceencoded by a nucleic acid sequence shown in FIGS. 1 a, 1 b or 1 c. Inother embodiments, the SRP amino acid homologs have sequence identityover at least 15 contiguous amino acid residues, more preferably atleast 25 contiguous amino acid residues, and most preferably at least 35contiguous amino acid residues of FIGS. 1 a, 1 b or 1 c.

In another preferred embodiment, an isolated nucleic acid homolog of theinvention comprises a nucleotide sequence which is at least about50-60%, preferably at least about 60-70%, more preferably at least about70-75%, 75-80%, 80-85%, 85-90% or 90-95%, and even more preferably atleast about 95%, 96%, 97%, 98%, 99% or more identical to a nucleotidesequence shown FIGS. 1 a, 1 b or 1 c, or to a portion comprising atleast 20, 30, 40, 50, 60 consecutive nucleotides thereof. The preferablelength of sequence comparison for nucleic acids is at least 75nucleotides, more preferably at least 100 nucleotides and mostpreferably the entire length of the coding region.

It is further preferred that the isolated nucleic acid homolog of theinvention encodes a SRP, or portion thereof, that is at least 85%identical to an amino acid sequence of FIGS. 1 a, 1 b or 1 c and thatfunctions as a modulator of an environmental stress response in a plant.In a more preferred embodiment, overexpression of the nucleic acidhomolog in a plant increases the tolerance of the plant to anenvironmental stress.

For the purposes of the invention, the percent sequence identity betweentwo nucleic acid or polypeptide sequences is determined using the VectorNTI 6.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda,Md. 20814). A gap opening penalty of 15 and a gap extension penalty of6.66 are used for determining the percent identity of two nucleic acids.A gap opening penalty of 10 and a gap extension penalty of 0.1 are usedfor determining the percent identity of two polypeptides. All otherparameters are set at the default settings. For purposes of a multiplealignment (Clustal W algorithm), the gap opening penalty is 10, and thegap extension penalty is 0.05 with blosum62 matrix. It is to beunderstood that for the purposes of determining sequence identity whencomparing a DNA sequence to an RNA sequence, a thymidine nucleotide isequivalent to a uracil nucleotide.

In another aspect, the invention provides an isolated nucleic acidcomprising a polynucleotide that hybridizes to the polynucleotide ofFIGS. 1 a, 1 b or 1 c under stringent conditions. More particularly, anisolated nucleic acid molecule of the invention is at least 15nucleotides in length and hybridizes under stringent conditions to thenucleic acid molecule comprising a nucleotide sequence of FIGS. 1 a, 1 bor 1 c. In other embodiments, the nucleic acid is at least 30, 50, 100,250 or more nucleotides in length. Preferably, an isolated nucleic acidhomolog of the invention comprises a nucleotide sequence whichhybridizes under highly stringent conditions to the nucleotide sequenceshown in FIGS. 1 a, 1 b or 1 c, and functions as a modulator of stresstolerance in a plant. In a further preferred embodiment, overexpressionof the isolated nucleic acid homolog in a plant increases a plant'stolerance to an environmental stress.

As used herein with regard to hybridization for DNA to DNA blot, theterm “stringent conditions” refers in one embodiment to hybridizationovernight at 60° C. in 10× Denharts solution, 6×SSC, 0.5% SDS and 100g/ml denatured salmon sperm DNA. Blots are washed sequentially at 62° C.for 30 minutes each time in 3×SSC/0.1% SDS, followed by 1×SSC/0.1% SDSand finally 0.1×SSC/0.1% SDS. As also used herein, “highly stringentconditions” refers to hybridization overnight at 65° C. in 10×Denhartssolution, 6×SSC, 0.5% SDS and 100 g/ml denatured salmon sperm DNA. Blotsare washed sequentially at 65° C. for 30 minutes each time in 3×SSC/0.1%SDS, followed by 1×SSC/0.1% SDS and finally 0.1×SSC/0.1% SDS. Methodsfor nucleic acid hybridizations are described in Meinkoth and Wahl,1984, Anal. Biochem. 138:267-284; Ausubel et al. eds, 1995, CurrentProtocols in Molecular Biology, Chapter 2, Greene Publishing andWiley-Interscience, New York; and Tijssen, 1993, Laboratory Techniquesin Biochemistry and Molecular Biology: Hybridization with Nucleic AcidProbes, Part I, Chapter 2, Elsevier, New York. Preferably, an isolatednucleic acid molecule of the invention that hybridizes under stringentor highly stringent conditions to a sequence of FIGS. 1 a, 1 b or 1 ccorresponds to a naturally occurring nucleic acid molecule. As usedherein, a “naturally occurring” nucleic acid molecule refers to an RNAor DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural polypeptide). In one embodiment, the nucleicacid encodes a naturally occurring Saccharomyces cerevisiae, E. coli,Brassica napus, Glycine max, or Oryza sativa SRP.

Using the above-described methods, and others known to those of skill inthe art, one of ordinary skill in the art can isolate homologs of theSRPs comprising amino acid sequences shown in FIGS. 1 a, 1 b or 1 c. Onesubset of these homologs are allelic variants. As used herein, the term“allelic variant” refers to a nucleotide sequence containingpolymorphisms that lead to changes in the amino acid sequences of a SRPand that exist within a natural population (e.g., a plant species orvariety). Such natural allelic variations can typically result in 1-5%variance in a SRP nucleic acid. Allelic variants can be identified bysequencing the nucleic acid sequence of interest in a number ofdifferent plants, which can be readily carried out by usinghybridization probes to identify the same SRP genetic locus in thoseplants. Any and all such nucleic acid variations and resulting aminoacid polymorphisms or variations in a SRP that are the result of naturalallelic variation and that do not alter the functional activity of aSRP, are intended to be within the scope of the invention.

An isolated nucleic acid molecule encoding a SRP having sequenceidentity with a polypeptide sequence of FIGS. 1 a, 1 b or 1 c can becreated by introducing one or more nucleotide substitutions, additionsor deletions into a nucleotide sequence of FIGS. 1 a, 1 b or 1 c,respectively, such that one or more amino acid substitutions, additions,or deletions are introduced into the encoded polypeptide. Mutations canbe introduced into one of the sequences of FIGS. 1 a, 1 b or 1 c bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain.

Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a SRP is preferablyreplaced with another amino acid residue from the same side chainfamily.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a SRP coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened for aSRP activity described herein to identify mutants that retain SRPactivity. Following mutagenesis of one of the sequences of theinvention, the encoded polypeptide can be expressed recombinantly andthe activity of the polypeptide can be determined by analyzing thestress tolerance of a plant expressing the polypeptide as describedherein.

Additionally, optimized SRP nucleic acids can be created. As usedherein, “optimized” refers to a nucleic acid that is geneticallyengineered to increase its expression in a given plant or animal. Toprovide plant optimized SRP nucleic acids, the DNA sequence of the genecan be modified to 1) comprise codons preferred by highly expressedplant genes; 2) comprise an A+T content in nucleotide base compositionto that substantially found in plants; 3) form a plant initiationsequence; or 4) eliminate sequences that cause destabilization,inappropriate polyadenylation, degradation, and termination of RNA, orthat form secondary structure hairpins or RNA splice sites. Increasedexpression of SRP nucleic acids in plants can be achieved by utilizingthe distribution frequency of codon usage in plants in general or aparticular plant. Methods for optimizing nucleic acid expression inplants can be found in EPA 0359472; EPA 0385962; PCT Application No. WO91/16432; U.S. Pat. No. 5,380,831; U.S. Pat. No. 5,436,391; Perlack etal., 1991, Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al.,1989, Nucleic Acids Res. 17:477-498.

As used herein, “frequency of preferred codon usage” refers to thepreference exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. To determine the frequency ofusage of a particular codon in a gene, the number of occurrences of thatcodon in the gene is divided by the total number of occurrences of allcodons specifying the same amino acid in the gene. Similarly, thefrequency of preferred codon usage exhibited by a host cell can becalculated by averaging frequency of preferred codon usage in a largenumber of genes expressed by the host cell. It is preferable that thisanalysis be limited to genes that are highly expressed by the host cell.The percent deviation of the frequency of preferred codon usage for asynthetic gene from that employed by a host cell is calculated first bydetermining the percent deviation of the frequency of usage of a singlecodon from that of the host cell followed by obtaining the averagedeviation over all codons. As defined herein, this calculation includesunique codons (i.e., ATG and TGG). In general terms, the overall averagedeviation of the codon usage of an optimized gene from that of a hostcell is calculated using the equation 1A=n=1ZX_(n)−Y_(n)X_(n) times 100Zwhere X_(n)=frequency of usage for codon n in the host cell;Y_(n)=frequency of usage for codon n in the synthetic gene; n representsan individual codon that specifies an amino acid; and the total numberof codons is Z. The overall deviation of the frequency of codon usage,A, for all amino acids should preferably be less than about 25%, andmore preferably less than about 10%.

Hence, a SRP nucleic acid can be optimized such that its distributionfrequency of codon usage deviates, preferably, no more than 25% fromthat of highly expressed plant genes and, more preferably, no more thanabout 10%. In addition, consideration is given to the percentage G+Ccontent of the degenerate third base (monocotyledons appear to favor G+Cin this position, whereas dicotyledons do not). It is also recognizedthat the XCG (where X is A, T, C, or G) nucleotide is the leastpreferred codon in dicots whereas the XTA codon is avoided in bothmonocots and dicots. Optimized SRP nucleic acids of this invention alsopreferably have CG and TA doublet avoidance indices closelyapproximating those of the chosen host plant (i.e., Brassica napus,Glycine max, or Oryza sativa). More preferably these indices deviatefrom that of the host by no more than about 10-15%.

In addition to the nucleic acid molecules encoding the SRPs describedabove, another aspect of the invention pertains to isolated nucleic acidmolecules that are antisense thereto. Antisense polynucleotides arethought to inhibit gene expression of a target polynucleotide byspecifically binding the target polynucleotide and interfering withtranscription, splicing, transport, translation, and/or stability of thetarget polynucleotide. Methods are described in the prior art fortargeting the antisense polynucleotide to the chromosomal DNA, to aprimary RNA transcript, or to a processed mRNA. Preferably, the targetregions include splice sites, translation initiation codons, translationtermination codons, and other sequences within the open reading frame.

The term “antisense,” for the purposes of the invention, refers to anucleic acid comprising a polynucleotide that is sufficientlycomplementary to all or a portion of a gene, primary transcript, orprocessed mRNA, so as to interfere with expression of the endogenousgene. “Complementary” polynucleotides are those that are capable of basepairing according to the standard Watson-Crick complementarity rules,bpecifically, purines will base pair with pyrimidines to form acombination of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. It is understood that twopolynucleotides may hybridize to each other even if they are notcompletely complementary to each other, provided that each has at leastone region that is substantially complementary to the other. The term“antisense nucleic acid” includes single stranded RNA as well asdouble-stranded DNA expression cassettes that can be transcribed toproduce an antisense RNA. “Active” antisense nucleic acids are antisenseRNA molecules that are capable of selectively hybridizing with a primarytranscript or mRNA encoding a polypeptide having at least 80% sequenceidentity with the polypeptide of FIGS. 1 a, 1 b or 1 c.

The antisense nucleic acid can be complementary to an entire SRP codingstrand, or to only a portion thereof. In one embodiment, an antisensenucleic acid molecule is antisense to a “coding region” of the codingstrand of a nucleotide sequence encoding a SRP. The term “coding region”refers to the region of the nucleotide sequence comprising codons thatare translated into amino acid residues. In another embodiment, theantisense nucleic acid molecule is antisense to a “noncoding region” ofthe coding strand of a nucleotide sequence encoding a SRP. The term“noncoding region” refers to 5′ and 3′ sequences that flank the codingregion that are not translated into amino acids (i.e., also referred toas 5′ and 3′ untranslated regions). The antisense nucleic acid moleculecan be complementary to the entire coding region of SRP mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of SRP mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of PKSRP mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. Typically, the antisense molecules of the presentinvention comprise an RNA having 60-100% sequence identity with at least14 consecutive nucleotides of one of the nucleic acid of FIGS. 1 a, 1 bor 1 c. Preferably, the sequence identity will be at least 70%, morepreferably at least 75%, 80%, 85%, 90%, 95%, 98% and most preferably99%.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al., 1987, Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., 1987, Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a SRP tothereby inhibit expression of the polypeptide, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic (includingplant) promoter are preferred.

As an alternative to antisense polynucleotides, ribozymes, sensepolynucleotides, or double stranded RNA (dsRNA) can be used to reduceexpression of a SRP polypeptide. By “ribozyme” is meant a catalyticRNA-based enzyme with ribonuclease activity which is capable of cleavinga single-stranded nucleic acid, such as an mRNA, to which it has acomplementary region. Ribozymes (e.g., hammerhead ribozymes described inHaselhoff and Gerlach, 1988, Nature 334:585-591) can be used tocatalytically cleave SRP mRNA transcripts to thereby inhibit translationof SRP mRNA. A ribozyme having specificity for a SRP-encoding nucleicacid can be designed based upon the nucleotide sequence of a SRP cDNA,as disclosed herein (i.e., FIGS. 1 a, 1 b or 1 c) or on the basis of aheterologous sequence to be isolated according to methods taught in thisinvention. For example, a derivative of a Tetrahymena L-19 IVS RNA canbe constructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in a SRP-encodingmRNA. See, e.g., U.S. Pat. Nos. 4,987,071 and 5,116,742 to Cech et al.Alternatively, SRP mRNA can be used to select a catalytic RNA having aspecific ribonuclease activity from a pool of RNA molecules. See, e.g.,Bartel, D. and Szostak, J. W., 1993, Science 261:1411-1418. In preferredembodiments, the ribozyme will contain a portion having at least 7, 8,9, 10, 12, 14, 16, 18 or 20 nucleotides, and more preferably 7 or 8nucleotides, that have 100% complementarity to a portion of the targetRNA. Methods for making ribozymes are known to those skilled in the art.See, e.g., U.S. Pat. Nos. 6,025,167; 5,773,260; and 5,496,698.

The term “dsRNA,” as used herein, refers to RNA hybrids comprising twostrands of RNA. The dsRNAs can be linear or circular in structure. In apreferred embodiment, dsRNA is specific for a polynucleotide encodingeither the polypeptide of FIGS. 1 a, 1 b or 1 c or a polypeptide havingat least 70% sequence identity with a polypeptide of FIGS. 1 a, 1 b or 1c. The hybridizing RNAs may be substantially or completelycomplementary. By “substantially complementary,” is meant that when thetwo hybridizing RNAs are optimally aligned using the BLAST program asdescribed above, the hybridizing portions are at least 95%complementary. Preferably, the dsRNA will be at least 100 base pairs inlength. Typically, the hybridizing RNAs will be of identical length withno over hanging 5′ or 3′ ends and no gaps. However, dsRNAs having 5′ or3′ overhangs of up to 100 nucleotides may be used in the methods of theinvention.

The dsRNA may comprise ribonucleotides or ribonucleotide analogs, suchas 2′-O-methyl ribosyl residues, or combinations thereof. See, e.g.,U.S. Pat. Nos. 4,130,641 and 4,024,222. A dsRNA polyriboinosinicacid:polyribocytidylic acid is described in U.S. Pat. No. 4,283,393.Methods for making and using dsRNA are known in the art. One methodcomprises the simultaneous transcription of two complementary DNAstrands, either in vivo, or in a single in vitro reaction mixture. See,e.g., U.S. Pat. No. 5,795,715. In one embodiment, dsRNA can beintroduced into a plant or plant cell directly by standardtransformation procedures. Alternatively, dsRNA can be expressed in aplant cell by transcribing two complementary RNAs.

Other methods for the inhibition of endogenous gene expression, such astriple helix formation (Moser et al., 1987, Science 238:645-650 andCooney et al., 1988, Science 241:456-459) and cosuppression (Napoli etal., 1990, The Plant Cell 2:279-289) are known in the art. Partial andfull-length cDNAs have been used for the cosuppression of endogenousplant genes. See, e.g., U.S. Pat. Nos. 4,801,340, 5,034,323, 5,231,020,and 5,283,184; Van der Kroll et al., 1990, The Plant Cell 2:291-299;Smith et al., 1990, Mol. Gen. Genetics 224:477-481 and Napoli et al.,1990, The Plant Cell 2:279-289.

For sense suppression, it is believed that introduction of a sensepolynucleotide blocks transcription of the corresponding target gene.The sense polynucleotide will have at least 65% sequence identity withthe target plant gene or RNA. Preferably, the percent identity is atleast 80%, 90%, 95% or more. The introduced sense polynucleotide neednot be full length relative to the target gene or transcript.Preferably, the sense polynucleotide will have at least 65% sequenceidentity with at least 100 consecutive nucleotides of one of the nucleicacids of FIGS. 1 a, 1 b or 1 c. The regions of identity can compriseintrons and/or exons and untranslated regions. The introduced sensepolynucleotide may be present in the plant cell transiently, or may bestably integrated into a plant chromosome or extrachromosomal replicon.

Moreover, nucleic acid molecules encoding SRP from the same or otherspecies such as SRP analogs, orthologs and paralogs, are intended to bewithin the scope of the present invention. As used herein, the term“analogs” refers to two nucleic acids that have the same or similarfunction, but that have evolved separately in unrelated organisms. Asused herein, the term “orthologs” refers to two nucleic acids fromdifferent species that have evolved from a common ancestral gene byspeciation. Normally, orthologs encode proteins having the same orsimilar functions. As also used herein, the term “paralogs” refers totwo nucleic acids that are related by duplication within a genome.Paralogs usually have different functions, but these functions may berelated (Tatusov, R. L. et al. 1997 Science 278(5338):631-637). Analogs,orthologs and paralogs of a naturally occurring stress related proteincan differ from the naturally occurring stress related protein bypost-translational modifications, by amino acid sequence differences, orby both. Post-translational modifications include in vivo and in vitrochemical derivatization of polypeptides e.g., acetylation,carboxylation, phosphorylation or glycosylation, and such modificationsmay occur during polypeptide synthesis or processing or followingtreatment with isolated modifying enzymes. In particular, orthologs ofthe invention will generally exhibit at least 80-85%, more preferably90%, 91%, 92%, 93%, 94%, and most preferably 95%, 96%, 97%, 98% or even99% identity or homology with all or part of a naturally occurringstress related protein amino acid sequence and will exhibit a functionsimilar to a stress related protein. Orthologs of the present inventionare also preferably capable of participating in the stress response inplants.

In addition to naturally-occurring variants of a stress related proteinsequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into anucleotide sequence of the FIGS. 1 a, 1 b or 1 c, thereby leading tochanges in the amino acid sequence of the encoded stress relatedprotein, without altering the functional ability of the stress relatedprotein or enhancing the functional ability of the stress relatedprotein. For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues can be made in asequence of FIG. 1. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of one of stress relatedproteins without altering the activity thereof, whereas an “essential”amino acid residue is required for stress related protein activity.Other amino acid residues, however, (e.g., those that are not conservedor only semi-conserved in the domain having SRP activity) may not beessential for activity and thus are likely to be amenable to alterationwithout altering SRP activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding stress related proteins that contain changes in aminoacid residues that are not essential for stress related proteinactivity. Such SRP differ in amino acid sequence from a sequence FIGS. 1a, 1 b or 1 c, yet retain at least one of the stress related proteinactivities described herein. In one embodiment, the isolated nucleicacid molecule comprises a nucleotide sequence encoding a protein,wherein the protein comprises an amino acid sequence at least about 50%homologous to an amino acid sequence of FIGS. 1 a, 1 b or 1 c.Preferably, the protein encoded by the nucleic acid molecule is at leastabout 50-60% homologous to one of the sequences of the FIGS. 1 a, 1 b or1 c, more preferably at least about 60-70% homologous to one of thesequences of the FIGS. 1 a, 1 b or 1 c, even more preferably at leastabout 70-80%, 80-90%, more preferably 90%, 91%, 92%, 93%, 94% homologousto one of the sequences of the FIGS. 1 a, 1 b or c and most preferablyat least about 96%, 97%, 98%, or 99% homologous to one of the sequencesof the FIGS. 1 a, 1 b or 1 c. The preferred stress related proteinhomologs of the present invention are preferably capable ofparticipating in the stress tolerance response in a plant. The homology(=identity) was calculated over the entire amino acid range. The programused was PileUp (J. Mol. Evolution., 25 (1987), 351-360, Higgins et al.,CABIOS, 5 1989: 151-153).

Homologs of the sequences given in FIGS. 1 a, 1 b or 1 c are furthermoreto be understood as meaning, for example, homologs, analogs, orthologsand paralogs which have at least 30% homology (=identity) at the derivedamino acid level, preferably at least 50%, 60%, 70% or 80% homology,especially preferably at least 85% homology, very especially preferably90% 91%, 92%, 93%, 94% homology, most preferably 95%, 96%, 97%, 98% or99% homology. The homology (=identity) was calculated over the entireamino acid range. The program used was PileUp (J. Mol. Evolution., 25(1987), 351-360, Higgens et al., CABIOS, 5 1989: 151-153) or the programGap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)and Smith and Waterman respectively (Adv. Appl. Math. 2; 482-489 (1981)]which are part of the GCG software package [Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711 (1991)]. The above mentionedpercentages of sequence homology are calculated with the program BestFitor Gap, preferably Gap, over the total sequence length with thefollowing parameters used: Gap Weight: 8, Length Weight: 2.

Variants shall also be encompassed, in particular, functional variantswhich can be obtained from the sequence shown in the FIGS. 1 a, 1 b or 1c by means of deletion, insertion or substitution of nucleotides, theenzymatic activity of the derived synthetic proteins being retained orenhanced.

An isolated nucleic acid molecule encoding a stress related proteinhomologous to a protein sequence of FIGS. 1 a, 1 b or 1 c can be createdby introducing one or more nucleotide substitutions, additions ordeletions into a nucleotide sequence of FIGS. 1 a, 1 b or 1 c such thatone or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced intoone of the sequences of FIGS. 1 a, 1 b or 1 c by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis. Anotherroute to the mutagenesis of enzymes, disclosed in the EuropeanPublication EP-A-0 909 821, is a method using the specific Escherichiacoli strain XL1-Red to generate mutants and altered the enzyme activity.

Preferably, conservative amino acid substitutions are made at one ormore predicted non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain.

Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a stress related protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a stress related protein codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for a stress related protein activity as describedherein to identify mutants that retain stress related protein activityor show enhanced stress related protein activity. Following mutagenesisof one of the sequences of the nucleic acid of FIGS. 1 a, 1 b or 1 c,the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined by analyzing the stress tolerance of aplant expressing the protein as described in the examples below.

A useful method to ascertain the level of transcription of the gene (anindicator of the amount of mRNA available for translation to the geneproduct) is to perform a Northern blot (for reference see, for example,Ausubel et al., 1988 Current Protocols in Molecular Biology, Wiley: NewYork). This information at least partially demonstrates the degree oftranscription of the gene. Total cellular RNA can be prepared fromcells, tissues or organs by several methods, all well-known in the art,such as that described in Bormann, E. R. et al., 1992 Mol. Microbiol.6:317-326. To assess the presence or relative quantity of proteintranslated from this mRNA, standard techniques, such as a Western blot,may be employed. These techniques are well known to one of ordinaryskill in the art (see, for example, Ausubel et al., 1988 CurrentProtocols in Molecular Biology, Wiley: New York).

The present invention also relates to a plant expression cassettecomprising a SRP coding nucleic acid selected from the group comprisingsequences of the nucleic acid of FIGS. 1 a, 1 b or 1 c and/or homologsor parts thereof operatively linked to regulatory sequences and/ortargeting sequences.

Further, object of the invention is an expression vector comprising aSRP encoding nucleic acid selected from the group comprising sequencesof the nucleic acid of FIGS. 1 a, 1 b or 1 c and/or homologs or partsthereof or a plant expression cassette as described above, wherebyexpression of the SRP coding nucleic acid in a host cell results inincreased tolerance to environmental stress, which is preferablyachieved by altering metabolic activity, as compared to a correspondingnon-transformed wild type host cell.

The invention further provides an isolated recombinant expression vectorcomprising a stress related protein encoding nucleic acid as describedabove, wherein expression of the vector or stress related proteinencoding nucleic acid, respectively in a host cell results in increasedtolerance and/or resistance to environmental stress, which is preferablyachieved by altering metabolic activity, as compared to thecorresponding non-transformed wild type of the host cell. As usedherein, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Further types of vectors can be linearizednucleic acid sequences, such as transposons, which are pieces of DNAwhich can copy and insert themselves. There have been 2 types oftransposons found: simple transposons, known as Insertion Sequences andcomposite transposons, which can have several genes as well as the genesthat are required for transposition.

Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

A plant expression cassette preferably contains regulatory sequencescapable of driving gene expression in plant cells and operably linked sothat each sequence can fulfill its function, for example, termination oftranscription by polyadenylation signals. Preferred polyadenylationsignals are those originating from Agrobacterium tumefaciens T-DNA suchas the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5(Gielen et al., 1984 EMBO J. 3:835) or functional equivalents thereofbut also all other terminators functionally active in plants aresuitable.

As plant gene expression is very often not limited on transcriptionallevels, a plant expression cassette preferably contains other operablylinked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al.,1987 Nucl. Acids Research 15:8693-8711).

Plant gene expression has to be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Preferred are promoters driving constitutive expression (Benfeyet al., 1989 EMBO J. 8:2195-2202) like those derived from plant viruseslike the 35S CaMV (Franck et al., 1980 Cell 21:285-294), the 19S CaMV(see also U.S. Pat. No. 5,352,605 and PCT Application No. WO 8402913) orplant promoters like those from Rubisco small subunit described in U.S.Pat. No. 4,962,028.

Additional advantageous regulatory sequences are, for example, includedin the plant promoters such as CaMV/35S [Franck et al., Cell 21 (1980)285-294], PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, OCS,lib4, usp, STLS1, B33, LEB4, nos or in the ubiquitin, napin or phaseolinpromoter. Also advantageous in this connection are inducible promoterssuch as the promoters described in EP-A-0 388 186 (benzyl sulfonamideinducible), Plant J. 2, 1992: 397-404 (Gatz et al., Tetracyclininducible), EP-A-0 335 528 (abscisic acid inducible) or WO 93/21334(ethanol or cyclohexenol inducible). Additional useful plant promotersare the cytosolic FBPase promotor or ST-LSI promoter of the potato(Stockhaus et al., EMBO J. 8, 1989, 2445), the phosphorybosylphyrophoshate amido transferase promoter of Glycine max (gene bankaccession No. U87999) or the noden specific promoter described in EP-A-0249 676. Additional particularly advantageous promoters are seedspecific promoters which can be used for monokotyledones ordikotyledones and are described in U.S. Pat. No. 5,608,152 (napinpromoter from rapeseed), WO 98/45461 (phaseolin promoter fromArobidopsis), U.S. Pat. No. 5,504,200 (phaseolin promoter from Phaseolusvulgaris), WO 91/13980 (Bce4 promoter from Brassica) and Baeumlein etal., Plant J., 2, 2, 1992: 233-239 (LEB4 promoter from leguminosa). Saidpromoters are useful in dikotyledones. The following promoters areuseful for example in monokotyledones Ipt-2- or Ipt-1- promoter frombarley (WO 95/15389 and WO 95/23230) or hordein promoter from barley.Other useful promoters are described in WO 99/16890.

It is possible in principle to use all natural promoters with theirregulatory sequences like those mentioned above for the novel process.It is also possible and advantageous in addition to use syntheticpromoters.

The gene construct may also comprise further genes which are to beinserted into the organisms and which are for example involved in stressresistance. It is possible and advantageous to insert and express inhost organisms regulatory genes such as genes for inducers, repressorsor enzymes which intervene by their enzymatic activity in theregulation, or one or more or all genes of a biosynthetic pathway. Thesegenes can be heterologous or homologous in origin. The inserted genesmay have their own promoter or else be under the control of samepromoter as the sequences of the nucleic acid of FIGS. 1 a, 1 b or 1 cor their homologs.

The gene construct advantageously comprises, for expression of the othergenes present, additionally 3′ and/or 5′ terminal regulatory sequencesto enhance expression, which are selected for optimal expressiondepending on the selected host organism and gene or genes.

These regulatory sequences are intended to make specific expression ofthe genes and protein expression possible as mentioned above. This maymean, depending on the host organism, for example that the gene isexpressed or overexpressed only after induction, or that it isimmediately expressed and/or overexpressed.

The regulatory sequences or factors may moreover preferably have abeneficial effect on expression of the introduced genes, and thusincrease it. It is possible in this way for the regulatory elements tobe enhanced advantageously at the transcription level by using strongtranscription signals such as promoters and/or enhancers. However, inaddition, it is also possible to enhance translation by, for example,improving the stability of the mRNA.

Other preferred sequences for use in plant gene expression cassettes aretargeting-sequences necessary to direct the gene product in itsappropriate cell compartment (for review see Kermode, 1996 Crit. Rev.Plant Sci. 15(4):285-423 and references cited therein) such as thevacuole, the nucleus, all types of plastids like amyloplasts,chloroplasts, chromoplasts, the extracellular space, mitochondria, theendoplasmic reticulum, oil bodies, peroxisomes and other compartments ofplant cells.

Plant gene expression can also be facilitated via an inducible promoter(for review see Gatz, 1997 Annu. Rev. Plant Physiol. Plant Mol. Biol.48:89-108). Chemically inducible promoters are especially suitable ifgene expression is wanted to occur in a time specific manner.

Table 1 lists several examples of promoters that may be used to regulatetranscription of the stress related protein nucleic acid codingsequences.

TABLE 1 Examples of Tissue-specific and Stress inducible promoters inplants Expression Reference Cor78- Cold, drought, Ishitani, et al.,Plant Cell 9: 1935-1949 (1997). salt, ABA, wounding- Yamaguchi-Shinozakiand Shinozaki, Plant Cell inducible 6: 251-264 (1994). Rci2A - Cold,Capel et al., Plant Physiol 115: 569-576 (1997) dehydration-inducibleRd22 - Drought, salt Yamaguchi-Shinozaki and Shinozaki, Mol Gen Genet238: 17-25 (1993). Cor15A - Cold, Baker at al., Plant Mol. Biol. 24:701-713 (1994). dehydration, ABA GH3- Auxin inducible Liu et al., PlantCell 6: 645-657 (1994) ARSK1-Root, salt Hwang and Goodman, Plant J 8:37-43 (1995). inducible PtxA - Root, salt GenBank accession X67427inducible SbHRGP3 - Root Ahn at al., Plant Cell 8: 1477-1490 (1998).specific KST1 - Guard cell Plesch et al., Plant Journal. 28(4): 455-64,(2001) specific KAT1 - Guard cell Plesch et al., Gene 249: 83-89 (2000)specific Nakamura et al., Plant Physiol. 109: 371-374 (1995) salicylicacid PCT Application No. WO 95/19443 inducible tetracycline inducibleGatz et al. Plant J. 2: 397-404 (1992) Ethanol inducible PCT ApplicationNo. WO 93/21334 pathogen inducible Ward et al., 1993 Plant. Mol. Biol.22: 361-366 PRP1 heat inducible hsp80 U.S. Pat. No. 5,187,267 coldinducible alpha- PCT Application No. WO 96/12814 amylase Wound-induciblepinII European Patent No. 375091 RD29A - salt- Yamaguchi-Shinozalei etal. (1993) Mol. Gen. inducible Genet. 236: 331-340 plastid-specificviral PCT Application No. WO 95/16783 and. WO RNA-polymerase 97/06250

Other promotors, e.g. superpromotor (Ni et al. “Plant Journal 7, 1995:661-676), Ubiquitin promotor (Callis et al., J. Biol. Chem., 1990, 265:12486-12493; U.S. Pat. No. 5,510,474; U.S. Pat. No. 6,020,190; Kawallecket al., Plant. Molecular Biology, 1993, 21: 673-684) or 34S promotor(GenBank Accession numbers M59930 and X16673) were similar useful forthe present invention and are known to a person skilled in the art.

Developmental stage-preferred promoters are preferentially expressed atcertain stages of development. Tissue and organ preferred promotersinclude those that are preferentially expressed in certain tissues ororgans, such as leaves, roots, seeds, or xylem. Examples of tissuepreferred and organ preferred promoters include, but are not limited tofruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred,integument-preferred, tuber-preferred, stalk-preferred,pericarp-preferred, and leaf-preferred, stigma-preferred,pollen-preferred, anther-preferred, a petal-preferred, sepal-preferred,pedicel-preferred, silique-preferred, stem-preferred, root-preferredpromoters, and the like. Seed preferred promoters are preferentiallyexpressed during seed development and/or germination. For example, seedpreferred promoters can be embryo-preferred, endosperm preferred, andseed coat-preferred. See Thompson et al., 1989, BioEssays 10:108.Examples of seed preferred promoters include, but are not limited to,cellulose synthase (celA), Ciml, gamma-zein, globulin-1, maize 19 kDzein (cZ19B1), and the like.

Other promoters useful in the expression cassettes of the inventioninclude, but are not limited to, the major chlorophyll a/b bindingprotein promoter, histone promoters, the Ap3 promoter, the β-conglycinpromoter, the napin promoter, the soybean lectin promoter, the maize 15kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter, theg-zein promoter, the waxy, shrunken 1, shrunken 2 and bronze promoters,the Zm13 promoter (U.S. Pat. No. 5,086,169), the maize polygalacturonasepromoters (PG) (U.S. Pat. Nos. 5,412,085 and 5,545,546), and the SGB6promoter (U.S. Pat. No. 5,470,359), as well as synthetic or othernatural promoters.

Additional flexibility in controlling heterologous gene expression inplants may be obtained by using DNA binding domains and responseelements from heterologous sources (i.e., DNA binding domains fromnon-plant sources). An example of such a heterologous DNA binding domainis the LexA DNA binding domain (Brent and Ptashne, 1985, Cell43:729-736).

The invention further provides a recombinant expression vectorcomprising a SRP DNA molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner that allowsfor expression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to a SRP mRNA. Regulatory sequences operatively linkedto a nucleic acid molecule cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types. For instance, viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific, or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid, or attenuated virus wherein antisensenucleic acids are produced under the control of a high efficiencyregulatory region. The activity of the regulatory region can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genes,see Weintraub, H. et al., 1986, Antisense RNA as a molecular tool forgenetic analysis, Reviews—Trends in Genetics, Vol. 1(1), and Mol et al.,1990, FEBS Letters 268:427-430.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but they also apply to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein. A host cell can be any prokaryotic or eukaryotic cell. Forexample, a SRP can be expressed in bacterial cells such as C.glutamicum, yeast, E. coli, insect cells, fungal cells, or mammaliancells (such as Chinese hamster ovary cells (CHO) or COS cells), algae,ciliates, plant cells, fungi, or other microorganisms like C.glutamicum. Other suitable host cells are known to those skilled in theart.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a SRP.Accordingly, the invention further provides methods for producing SRPsusing the host cells of the invention. In one embodiment, the methodcomprises culturing the host cell of invention (into which a recombinantexpression vector encoding a SRP has been introduced, or into whichgenome has been introduced a gene encoding a wild-type or altered SRP)in a suitable medium until SRP is produced. In another embodiment, themethod further comprises isolating SRPs from the medium or the hostcell.

Another aspect of the invention pertains to isolated SRPs, andbiologically active portions thereof. An “isolated” or “purified”polypeptide or biologically active portion thereof is free of some ofthe cellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof SRP in which the polypeptide is separated from some of the cellularcomponents of the cells in which it is naturally or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of a SRP having less than about30% (by dry weight) of non-SRP material (also referred to herein as a“contaminating polypeptide”), more preferably less than about 20% ofnon-SRP material, still more preferably less than about 10% of non-SRPmaterial, and most preferably less than about 5% non-PKSRP material.

When the SRP or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the polypeptide preparation. The language “substantially freeof chemical precursors or other chemicals” includes preparations of SRPin which the polypeptide is separated from chemical precursors or otherchemicals that are involved in the synthesis of the polypeptide. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of a SRP having less than about30% (by dry weight) of chemical precursors or non-SRP chemicals, morepreferably less than about 20% chemical precursors or non-SRP chemicals,still more preferably less than about 10% chemical precursors or non-SRPchemicals, and most preferably less than about 5% chemical precursors ornon-SRP chemicals. In preferred embodiments, isolated polypeptides, orbiologically active portions thereof, lack contaminating polypeptidesfrom the same organism from which the SRP is derived. Typically, suchpolypeptides are produced by recombinant expression of, for example, aSaccharomyces cerevisiae, E. coli, Brassica napus, Glycine max, or Oryzasativa SRP in plants other than Saccharomyces cerevisiae, E. coli,Brassica napus, Glycine max, or Oryza sativa, or microorganisms such asC. glutamicum, ciliates, algae or fungi.

The nucleic acid molecules, polypeptides, polypeptide homologs, fusionpolypeptides, primers, vectors, and host cells described herein can beused in one or more of the following methods: identification ofSaccharomyces cerevisiae, E. coli, Brassica napus, Glycine max, or Oryzasativa and related organisms; mapping of genomes of organisms related toSaccharomyces cerevisiae, E. coli, Brassica napus, Glycine max, or Oryzasativa; identification and localization of Saccharomyces cerevisiae, E.coli, Brassica napus, Glycine max, or Oryza sativa sequences ofinterest; evolutionary studies; determination of SRP regions requiredfor function; modulation of a SRP activity; modulation of the metabolismof one or more cell functions; modulation of the transmembrane transportof one or more compounds; modulation of stress resistance; andmodulation of expression of SRP nucleic acids.

The SRP nucleic acid molecules of the invention are also useful forevolutionary and polypeptide structural studies. The metabolic andtransport processes in which the molecules of the invention participateare utilized by a wide variety of prokaryotic and eukaryotic cells; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment of which regions of the sequenceare conserved and which are not, which may aid in determining thoseregions of the polypeptide that are essential for the functioning of theenzyme. This type of determination is of value for polypeptideengineering studies and may give an indication of what the polypeptidecan tolerate in terms of mutagenesis without losing function.

Manipulation of the SRP nucleic acid molecules of the invention mayresult in the production of SRPs having functional differences from thewild-type SRPs. These polypeptides may be improved in efficiency oractivity, may be present in greater numbers in the cell than is usual,or may be decreased in efficiency or activity.

There are a number of mechanisms by which the alteration of a SRP of theinvention may directly affect stress response and/or stress tolerance.In the case of plants expressing SRPs, increased transport can lead toimproved salt and/or solute partitioning within the plant tissue andorgans. By either increasing the number or the activity of transportermolecules which exportionic molecules from the cell, it may be possibleto affect the salt tolerance of the cell.

The effect of the genetic modification in plants, C. glutamicum, fungi,algae, or ciliates on stress tolerance can be assessed by growing themodified microorganism or plant under less than suitable conditions andthen analyzing the growth characteristics and/or metabolism of theplant. Such analysis techniques are well known to one skilled in theart, and include dry weight, wet weight, polypeptide synthesis,carbohydrate synthesis, lipid synthesis, evapotranspiration rates,general plant and/or crop yield, flowering, reproduction, seed setting,root growth, respiration rates, photosynthesis rates, etc. (Applicationsof HPLC in Biochemistry in: Laboratory Techniques in Biochemistry andMolecular Biology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3,Chapter III: Product recovery and purification, page 469-714, VCH:Weinheim; Belter, P. A. et al., 1988, Bioseparations: downstreamprocessing for biotechnology, John Wiley and Sons; Kennedy, J. F. andCabral, J. M. S., 1992, Recovery processes for biological materials,John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D., 1988,Biochemical separations, in: Ulmann's Encyclopedia of IndustrialChemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J., 1989, Separation and purification techniques in biotechnology, NoyesPublications).

For example, yeast expression vectors comprising the nucleic acidsdisclosed herein, or fragments thereof, can be constructed andtransformed into Saccharomyces cerevisiae using standard protocols. Theresulting transgenic cells can then be assayed for fail or alteration oftheir tolerance to drought, salt, and temperature stress. Similarly,plant expression vectors comprising the nucleic acids disclosed herein,or fragments thereof, can be constructed and transformed into anappropriate plant cell such as Arabidopsis, soy, rape, maize, wheat,Medicago truncatula, etc., using standard protocols. The resultingtransgenic cells and/or plants derived therefrom can then be assayed forfail or alteration of their tolerance to drought, salt, temperaturestress, and lodging.

The engineering of one or more SRP genes of the invention may alsoresult in SRPs having altered activities which indirectly impact thestress response and/or stress tolerance of algae, plants, ciliates, orfungi, or other microorganisms like C. glutamicum. For example, thenormal biochemical processes of metabolism result in the production of avariety of products (e.g., hydrogen peroxide and other reactive oxygenspecies) which may actively interfere with these same metabolicprocesses. For example, peroxynitrite is known to nitrate tyrosine sidechains, thereby inactivating some enzymes having tyrosine in the activesite (Groves, J. T., 1999, Curr. Opin. Chem. Biol. 3(2):226-235). Whilethese products are typically excreted, cells can be genetically alteredto transport more products than is typical for a wild-type cell. Byoptimizing the activity of one or more PKSRPs of the invention which areinvolved in the export of specific molecules, such as salt molecules, itmay be possible to improve the stress tolerance of the cell.

Additionally, the sequences disclosed herein, or fragments thereof, canbe used to generate knockout mutations in the genomes of variousorganisms, such as bacteria, mammalian cells, yeast cells, and plantcells (Girke, T., 1998, The Plant Journal 15:39-48). The resultantknockout cells can then be evaluated for their ability or capacity totolerate various stress conditions, their response to various stressconditions, and the effect on the phenotype and/or genotype of themutation. For other methods of gene inactivation, see U.S. Pat. No.6,004,804 “Non-Chimeric Mutational Vectors” and Puttaraju et al., 1999,Spliceosome-mediated RNA trans-splicing as a tool for gene therapy,Nature Biotechnology 17:246-252.

The aforementioned mutagenesis strategies for SRPs resulting inincreased stress resistance are not meant to be limiting; variations onthese strategies will be readily apparent to one skilled in the art.Using such strategies, and incorporating the mechanisms disclosedherein, the nucleic acid and polypeptide molecules of the invention maybe utilized to generate algae, ciliates, plants, fungi, or othermicroorganisms like C. glutamicum expressing mutated PKSRP nucleic acidand polypeptide molecules such that the stress tolerance is improved.

The present invention also provides antibodies that specifically bind toa SRP, or a portion thereof, as encoded by a nucleic acid describedherein. Antibodies can be made by many well-known methods (See, e.g.Harlow and Lane, “Antibodies; A Laboratory Manual,” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigencan be injected into an animal in an amount and in intervals sufficientto elicit an immune response. Antibodies can either be purifieddirectly, or spleen cells can be obtained from the animal. The cells canthen fused with an immortal cell line and screened for antibodysecretion. The antibodies can be used to screen nucleic acid clonelibraries for cells secreting the antigen. Those positive clones canthen be sequenced. See, for example, Kelly et al., 1992, Bio/Technology10:163-167; Bebbington et al., 1992, Bio/Technology 10:169-175.

The phrases “selectively binds” and “specifically binds” with thepolypeptide refer to a binding reaction that is determinative of thepresence of the polypeptide in a heterogeneous population ofpolypeptides and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bound to a particular polypeptidedo not bind in a significant amount to other polypeptides present in thesample. Selective binding of an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular polypeptide. A variety of immunoassay formats may be used toselect antibodies that selectively bind with a particular polypeptide.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies selectively immunoreactive with a polypeptide. See Harlow andLane, “Antibodies, A Laboratory Manual,” Cold Spring HarborPublications, New York, (1988), for a description of immunoassay formatsand conditions that could be used to determine selective binding.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious hosts. A description of techniques for preparing such monoclonalantibodies may be found in Stites et al., eds., “Basic and ClinicalImmunology,” (Lange Medical Publications, Los Altos, Calif., FourthEdition) and references cited therein, and in Harlow and Lane,“Antibodies, A Laboratory Manual,” Cold Spring Harbor Publications, NewYork, (1988).

Gene expression in plants is regulated by the interaction of proteintranscription factors with specific nucleotide sequences within theregulatory region of a gene. A common type of transcription factorcontains zinc finger (ZF) motifs. Each ZF module is approximately 30amino acids long folded around a zinc ion. The DNA recognition domain ofa ZF protein is a α-helical structure that inserts into the major groveof the DNA double helix. The module contains three amino acids that bindto the DNA with each amino acid contacting a single base pair in thetarget DNA sequence. ZF motifs are arranged in a modular repeatingfashion to form a set of fingers that recognize a contiguous DNAsequence. For example, a three-fingered ZF motif will recognize 9 bp ofDNA. Hundreds of proteins have been shown to contain ZF motifs withbetween 2 and 37 ZF modules in each protein (Isalan M, et al., 1998Biochemistry 37(35):12026-33; Moore M, et al., 2001 Proc. Natl. Acad.Sci. USA 98(4):1432-1436 and 1437-1441; U.S. Pat. Nos. 6,007,988 and6,013,453).

The regulatory region of a plant gene contains many short DNA sequences(cis-acting elements) that serve as recognition domains fortranscription factors, including ZF proteins. Similar recognitiondomains in different genes allow the coordinate expression of severalgenes encoding enzymes in a metabolic pathway by common transcriptionfactors. Variation in the recognition domains among members of a genefamily facilitates differences in gene expression within the same genefamily, for example, among tissues and stages of development and inresponse to environmental conditions.

Typical ZF proteins contain not only a DNA recognition domain but also afunctional domain that enables the ZF protein to activate or represstranscription of a specific gene. Experimentally, an activation domainhas been used to activate transcription of the target gene (U.S. Pat.No. 5,789,538 and patent application WO9519431), but it is also possibleto link a transcription repressor domain to the ZF and thereby inhibittranscription (patent applications WO00/47754 and WO2001002019). It hasbeen reported that an enzymatic function such as nucleic acid cleavagecan be linked to the ZF (patent application WO00/20622)

The invention provides a method that allows one skilled in the art toisolate the regulatory region of one or more stress related proteinencoding genes from the genome of a plant cell and to design zinc fingertranscription factors linked to a functional domain that will interactwith the regulatory region of the gene. The interaction of the zincfinger protein with the plant gene can be designed in such a manner asto alter expression of the gene and preferably thereby alter metabolicactivity to confer increased (or decreased) tolerance of abiotic stresssuch as drought. The invention provides a method of producing atransgenic plant with a transgene encoding this designed transcriptionfactor, or alternatively a natural transcription factor, that modifiestranscription of the Stress-Related Protein, particularly stress relatedprotein gene to provide increased tolerance of environmental stress,which is preferably achieved by altering metabolic activity. Such aregulation of plant genes by artificial polydactyl zinc fingers has beendemonstrated by Ordiz et al. (Regulation of transgene Expression inplants with polydactyl zinc finger transcription factors, Ordiz et al.,PNAS, 99 (20) 13290-13295, 2002) or Guan et al. (Hertiable endogenousgene regulation in plants with designed polydactyl zinc fingertranscription factors, PNAS, Vol. 99 (20), 13296-13301 (2002)).

In particular, the invention provides a method of producing a transgenicplant with a stress related protein coding nucleic acid, whereinexpression of the nucleic acid(s) in the plant results in increasedtolerance to environmental stress, which is preferably achieved byaltering metabolic activity, as compared to a wild type plantcomprising: (a) transforming a plant cell with an expression vectorcomprising a stress related protein encoding nucleic acid, and (b)generating from the plant cell a transgenic plant with an increasedtolerance to environmental stress as compared to a wild type plant. Forsuch plant transformation, binary vectors such as pBinAR can be used(Höfgen and Willmitzer, 1990 Plant Science 66:221-230). Moreoversuitable binary vectors are for example pBIN19, pBI101, pGPTV or pPZP(Hajukiewicz, P. et al., 1994, Plant Mol. Biol., 25: 989-994).

Construction of the binary vectors can be performed by ligation of thecDNA into the T-DNA. 5′ to the cDNA a plant promoter activatestranscription of the cDNA. A polyadenylation sequence is located 3′ tothe cDNA. Tissue-specific expression can be achieved by using a tissuespecific promoter as listed above. Also, any other promoter element canbe used. For constitutive expression within the whole plant, the CaMV35S promoter can be used. The expressed protein can be targeted to acellular compartment using a signal peptide, for example for plastids,mitochondria or endoplasmic reticulum (Kermode, 1996 Crit. Rev. PlantSci. 4(15):285-423). The signal peptide is cloned 5′ in frame to thecDNA to archive subcellular localization of the fusion protein.Additionally, promoters that are responsive to abiotic stresses can beused with, such as the Arabidopsis promoter RD29A. One skilled in theart will recognize that the promoter used should be operatively linkedto the nucleic acid such that the promoter causes transcription of thenucleic acid which results in the synthesis of a mRNA which encodes apolypeptide.

Alternate methods of transfection include the direct transfer of DNAinto developing flowers via electroporation or Agrobacterium mediatedgene transfer. Agrobacterium mediated plant transformation can beperformed using for example the GV3101(pMP90) (Koncz and Schell, 1986Mol. Gen. Genet. 204:383-396) or LBA4404 (Ooms et al., Plasmid, 1982, 7:15-29; Hoekema et al., Nature, 1983, 303: 179-180) Agrobacteriumtumefaciens strain. Transformation can be performed by standardtransformation and regeneration techniques (Deblaere et al., 1994 Nucl.Acids. Res. 13:4777-4788; Gelvin and Schilperoort, Plant MolecularBiology Manual, 2nd Ed.—Dordrecht: Kluwer Academic Publ., 1995.—inSect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, B Rand Thompson, J E, Methods in Plant Molecular Biology and Biotechnology,Boca Raton: CRC Press, 1993. —360 S., ISBN 0-8493-5164-2). For example,rapeseed can be transformed via cotyledon or hypocotyl transformation(Moloney et al., 1989 Plant Cell Reports 8:238-242; De Block et al.,1989 Plant Physiol. 91:694-701). Use of antibiotics for Agrobacteriumand plant selection depends on the binary vector and the Agrobacteriumstrain used for transformation. Rapeseed selection is normally performedusing kanamycin as selectable plant marker. Agrobacterium mediated genetransfer to flax can be performed using, for example, a techniquedescribed by Mlynarova et al., 1994 Plant Cell Report 13:282-285.Additionally, transformation of soybean can be performed using forexample a technique described in European Patent No. 0424 047, U.S. Pat.No. 5,322,783, European Patent No. 0397 687, U.S. Pat. No. 5,376,543 orU.S. Pat. No. 5,169,770. Transformation of maize can be achieved byparticle bombardment, polyethylene glycol mediated DNA uptake or via thesilicon carbide fiber technique (see, for example, Freeling and Walbot“The maize handbook” Springer Verlag: New York (1993) ISBN3-540-97826-7). A specific example of maize transformation is found inU.S. Pat. No. 5,990,387 and a specific example of wheat transformationcan be found in PCT Application No. WO 93/07256.

The stress related protein encoding nucleic acid molecules of theinvention have a variety of uses. Most importantly, the nucleic acid andamino acid sequences of the present invention can be used to transformplant cells or plants, thereby inducing tolerance to stresses such asdrought, high salinity and cold. The present invention thereforeprovides a transgenic plant transformed by a stress related proteinencoding nucleic acid (coding or antisense), wherein expression of thenucleic acid sequence in the plant results in increased tolerance toenvironmental stress as compared to a wild type plant. The increasedstress tolerance is apparent as an increase in the yield or quality ofthe plant. The transgenic plant can be a monocot or a dicot or agymnosperm plant. The invention further provides that the transgenicplant can be selected from maize, wheat, rye, oat, triticale, rice,barley, soybean, peanut, cotton, borage, sufflower, linseed, primrose,rapeseed, canola and turnip rape, manihot, pepper, sunflower, tagetes,solanaceous plant such as potato, tobacco, eggplant and tomato, Viciaspecies, pea, alfalfa, bushy plants such as coffee, cacao, tea, Salixspecies, trees such as oil palm, coconut, perennial grass, such asryegrass and fescue, and forage crops, such as alfalfa and clover andArabidopsis thaliana. Further the transgenic plant can be selected fromspruce, pine or fir for example.

In particular, the present invention describes using the expression ofstress related proteins to engineer drought-tolerant, salt-tolerantand/or cold-tolerant plants. This strategy has herein been demonstratedfor Arabidopsis thaliana, Ryegrass, Alfalfa, Rapeseed/Canola, Soybean,Corn and Wheat but its application is not restricted to these plants.Accordingly, the invention provides a transgenic plant containing astress related protein encoding gene selected from the nucleic acid ofFIGS. 1 a, 1 b or 1 c and/or homologs of the afore mentioned sequences,wherein the environmental stress is drought, increased salt or decreasedor increased temperature but its application is not restricted to theseadverse environments. Protection against other adverse conditions suchas heat, air pollution, heavy metals and chemical toxicants, forexample, may be obtained. In preferred embodiments, the environmentalstress is drought.

The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a stressrelated protein encoding gene in the plant. The invention provides thatthis method can be performed such that the stress tolerance isincreased. This can for example be done by the use of transcriptionfactors. In particular, the present invention provides methods ofproducing a transgenic plant having an increased tolerance toenvironmental stress as compared to a wild type plant due to increasedexpression of a stress related protein in the plant.

Growing the modified plants under stress conditions and then screeningand analyzing the growth characteristics and/or metabolic activityassess the effect of the genetic modification in plants on stresstolerance and/or resistance. Such analysis techniques are well known toone skilled in the art. They include next to screening (Römpp LexikonBiotechnologie, Stuttgart/New York: Georg Thieme Verlag 1992,“screening” p. 701) dry weight, wet weight, protein synthesis,carbohydrate synthesis, lipid synthesis, evapotranspiration rates,general plant and/or crop yield, flowering, reproduction, seed setting,root growth, respiration rates, photosynthesis rates, etc. (Applicationsof HPLC in Biochemistry in: Laboratory Techniques in Biochemistry andMolecular Biology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3,Chapter III: Product recovery and purification, page 469-714, VCH:Weinheim; Belter, P. A. et al., 1988 Bioseparations: downstreamprocessing for biotechnology, John Wiley and Sons; Kennedy, J. F. andCabral, J. M. S., 1992 Recovery processes for biological materials, JohnWiley and Sons; Shaeiwitz, J. A. and Henry, J. D., 1988 Biochemicalseparations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol. B3,Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F. J. (1989)Separation and purification techniques in biotechnology, NoyesPublications).

The engineering of one or more stress related protein encoding genes ofthe invention may also result in stress related proteins having alteredactivities which indirectly impact the stress response and/or stresstolerance of plants. For example, the normal biochemical processes ofmetabolism result in the production of a variety of products (e.g.,hydrogen peroxide and other reactive oxygen species) which may activelyinterfere with these same metabolic processes (for example,peroxynitrite is known to react with tyrosine side chains, therebyinactivating some enzymes having tyrosine in the active site (Groves, J.T., 1999 Curr. Opin. Chem. Biol. 3(2):226-235). By optimizing theactivity of one or more stress related proteins (enzymes) of theinvention, it may be possible to improve the stress tolerance of thecell.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes andvariations may be made therein without departing from the scope of theinvention. The invention is further illustrated by the followingexamples, which are not to be construed in any way as limiting. On thecontrary, it is to be clearly understood that various other embodiments,modifications and equivalents thereof, which, after reading thedescription herein, may suggest themselves to those skilled in the artwithout departing from the spirit of the present invention and/or thescope of the claims.

The invention also pertains to the use of SRP encoding nucleic acidselected form the group comprising the nucleic acid of FIGS. 1 a, 1 b or1 c and/or homologs of the afore mentioned sequences for preparing aplant cell with increased environmental stress tolerance, which ispreferably achieved by altering metabolic activity. The said sequencescan also be used for preparing a plant with increased environmentalstress tolerance.

Object of the invention is further the use of altered metabolic activityand/or a SRP encoding nucleic acid selected form the group of sequencesof the nucleic acid of FIGS. 1 a, 1 b or 1 c and/or homologs of theafore mentioned sequences or parts thereof as markers for selection ofplants with increased tolerance to environmental stress or as markersfor detection of stress in plants or plant cells.

Example 1 Engineering Stress-Tolerant Arabidopsis Plants byOver-Expressing Stress Related Protein Genes

Gene Cloning and Transformation of Arabidopsis Thaliana

Amplification

The standard protocol of Pfu DNA polymerase or a Pfu/Taq DNA polymerasemix (Herculase) was used for the amplification procedure. Amplified ORFfragments were analysed by gel electrophoresis. Each primer consists ofa universal 5′ end and ORF specific 3′ end whereby the universalsequences differ for the forward and reverse primers (forward primersequence contains an EcoRI for yeast or SmaI for E. coli and the reverseprimer sequence a SmaI for yeast or SacI for E. coli restriction site)allowing generally a unidirectional cloning success.

Amplification using the protocol of Pfu or Herculase DNA polymerase(Stratagene). Conditions: 1×PCR buffer, 0.2 mM dNTP, 100 ng genomic DNASaccharomyces cerevisiae (S288C) or 60 ng genomic DNA Escherichia coliK-12 (MG1655), 25 μmol forward primer, 25 μmol reverse primer, 2.5 u Pfuor Herculase DNA polymerase. 1st cycle for 3′ for yeast of 2′ for E.coli at 94° C., followed by 25 cycles for 30″ at 94° C., 30″ 55° C. foryeast or 60° C. for E. coli and 5-6′ 72° C., followed by 1 cycle for610′ at 72° C., final for 4° C. at ∞.

TABLE 2 Forward and reverse primer sequences used for ORF amplificationGene Forward Seq YGL263WGGAATTCCAGCTGACCACCATGGATGGAGCCAAATTTGAAAATAC (SEQ ID NO: 561) YGR004WGGAATTCCAGCTGACCACCATGAGCGAAATAAATAATGAAAATCTAG (SEQ ID NO: 562) YGR014WGGAATTCCAGCTGACCACCATGCAGTTTCCATTCGCTTGTCTC (SEQ ID NO: 563) YGL239CGGAATTCCAGCTGACCACCATGAAATTTTTGAAGAACAAAGCACC (SEQ ID NO: 564) YBL060WGGAATTCCAGCTGACCACCATGTGCGCCAGTTTAAACGAGGTA (SEQ ID NO: 565) YGL166WGGAATTCCAGCTGACCACCATGGTCGTAATTAACGGGGTCAAAT (SEQ ID NO: 566) YDL202WGGAATTCCAGCTGACCACCATGTTGCAGCTAAGGTTTATGCCT (SEQ ID NO: 567) YAL046CGGAATTCCAGCTGACCACCATGAAGCTCCCACAGACCATGCT (SEQ ID NO: 568) YDR101CGGAATTCCAGCTGACCACCATGGCTCTAGCTATCTCCCACGA (SEQ ID NO: 569) YDR108WGGAATTCCAGCTGACCACCATGGTTTTTTCTTATGAGCACTATATG (SEQ ID NO: 570) YAL064WGGAATTCCAGCTGACCACCATGCGATATACTGCAACTTTTCGG (SEQ ID NO: 571) YDR134CGGAATTCCAGCTGACCACCATGCAATTCTCTACCGTCGCTTCT (SEQ ID NO: 572) YFL031wGGAATTCCAGCTGACCACCATGAATAGCGAGTACGATTACCTGT (SEQ ID NO: 573) YFL052WGGAATTCCAGCTGACCACCATGGCCCGCAATAGACAAGCGT (SEQ ID NO: 574) YFL042CGGAATTCCAGCTGACCACCATGTCGGATGTAGATAACTGGGAA (SEQ ID NO: 575) YBR025CGGAATTCCAGCTGACCACCATGCCTCCAAAGAAGCAAGTCGAA (SEQ ID NO: 576) YER174CGGAATTCCAGCTGACCACCATGACTGTGGTTGAAATAAAAAGCC (SEQ ID NO: 577) YBR051WGGAATTCCAGCTGACCACCATGCACATTCTTTTCTTGTTTATTTTC (SEQ ID NO: 578) YER175CGGAATTCCAGCTGACCACCATGTCTACCTTTTCTGCTTCTGATT (SEQ ID NO: 579) YDR521WGGAATTCCAGCTGACCACCATGAAAGGTTCAAAATCGCACCTTG (SEQ ID NO: 580) YER167WGGAATTCCAGCTGACCACCATGCCGAAGAATAGTCACCACCAT (SEQ ID NO: 581) YER123WGGAATTCCAGCTGACCACCATGTCCCAACGATCTTCACAACAC (SEQ ID NO: 582) YDR415CGGAATTCCAGCTGACCACCATGGCCGATGAGGAACGTTTAAAG (SEQ ID NO: 583) YEL052WGGAATTCCAGCTGACCACCATGATCGCTTTGAAGCCCAATGCT (SEQ ID NO: 584) YDR536WGGAATTCCAGCTGACCACCATGAAGGATTTAAAATTATCGAATTTCA (SEQ ID NO: 585) YDR513WGGAATTCCAGCTGACCACCATGGAGACCAATTTTTCCTTCGACT (SEQ ID NO: 586) YEL045CGGAATTCCAGCTGACCACCATGAAATGTCACGCGAAACGGAC (SEQ ID NO: 587) YEL041WGGAATTCCAGCTGACCACCATGAAAACTGATAGATTACTGATTAAC (SEQ ID NO: 588) YDL238CGGAATTCCAGCTGACCACCATGACAAAAAGTGATTTATTATTTGATAA (SEQ ID NO: 589)YBR282W GGAATTCCAGCTGACCACCATGAAAGGCTCACCCATTTCTCAAT (SEQ ID NO: 590)YBR258C GGAATTCCAGCTGACCACCATGGCGTATAATCAAGAAGATAGTAA (SEQ ID NO: 591)YCL001W-AGGAATTCCAGCTGACCACCATGACCTTTTTACAATTTATCAATAATAATA (SEQ ID NO: 592)YBR274W GGAATTCCAGCTGACCACCATGAGTCTCTCGCAGGTGTCAC (SEQ ID NO: 593)YHR090C GGAATTCCAGCTGACCACCATGGATCCAAGTTTAGTTTTAGAGC (SEQ ID NO: 594)YGR121C GGAATTCCAGCTGACCACCATGGAGAGTCGAACTACAGGGC (SEQ ID NO: 595)YGR127W GGAATTCCAGCTGACCACCATGTGCATTTTAATGGCCACAAGG (SEQ ID NO: 596)YGR150C GGAATTCCAGCTGACCACCATGTACATGGCCAGATGTGGCC (SEQ ID NO: 597)YKL037W GGAATTCCAGCTGACCACCATGCAGACAATGGGCGGGGAG (SEQ ID NO: 598)YKL051W GGAATTCCAGCTGACCACCATGATTCAATTTAAAAGTCCAGGTAAC (SEQ ID NO: 599)YKL120W GGAATTCCAGCTGACCACCATGTCATCTGACAACTCTAAACAAG (SEQ ID NO: 600)YKL011C GGAATTCCAGCTGACCACCATGTCGACAGCACAGAAAGCTAAG (SEQ ID NO: 601)YKL017C GGAATTCCAGCTGACCACCATGAACAAAGAATTGGCTTCTAAGTT (SEQ ID NO: 602)YKL049C GGAATTCCAGCTGACCACCATGGAAACTGAAGTACCTGCACCA (SEQ ID NO: 603)YKL132C GGAATTCCAGCTGACCACCATGGATGATATAAGCGGAAGGCAAA (SEQ ID NO: 604)YGR126W GGAATTCCAGCTGACCACCATGCCTGTCCCATCTGTTACTGT (SEQ ID NO: 605)YKL070W GGAATTCCAGCTGACCACCATGTACATTCCTAAACATTTTGAGTC (SEQ ID NO: 606)YKL058W GGAATTCCAGCTGACCACCATGGCAGTACCCGGGTATTACGA (SEQ ID NO: 607)YHR130C GGAATTCCAGCTGACCACCATGACTAAAAGTATATATATTATCATCG (SEQ ID NO: 608)YIL070C GGAATTCCAGCTGACCACCATGTTCTTAAGAAGCGTTAACCGTG (SEQ ID NO: 609)YHR195W GGAATTCCAGCTGACCACCATGACTCGTCCCCCATTGGTTC (SEQ ID NO: 610)YIR022W GGAATTCCAGCTGACCACCATGAATCTAAGATTTGAATTGCAGAAA (SEQ ID NO: 611)YJL089W GGAATTCCAGCTGACCACCATGGCCAAGAGGAAATATGGCAG (SEQ ID NO: 612)YJL172W GGAATTCCAGCTGACCACCATGATCGCCTTACCAGTAGAGAAG (SEQ ID NO: 613)YHR113W GGAATTCCAGCTGACCACCATGTTCAGGATACAACTGAGAACTA (SEQ ID NO: 614)YHR175W GGAATTCCAGCTGACCACCATGGATGATAAGAAAACATGGAGTAC (SEQ ID NO: 615)YGR212W GGAATTCCAGCTGACCACCATGAATCTTAAACTTTCTGCTATTGAA (SEQ ID NO: 616)YJL024C GGAATTCCAGCTGACCACCATGATTCATGCAGTTCTAATATGTATG (SEQ ID NO: 617)YGR180c GGAATTCCAGCTGACCACCATGGAAGCACATAACCAATTTTTGAA (SEQ ID NO: 618)YJL179W GGAATTCCAGCTGACCACCATGTCACAGATAGCACAAGAAATGA (SEQ ID NO: 619)YJL001W GGAATTCCAGCTGACCACCATGAATGGAATTCAAGTGGACATCA (SEQ ID NO: 620)YJL208C GGAATTCCAGCTGACCACCATGTGCAGTAGGATACTCTTGTCC (SEQ ID NO: 621)YJL152W GGAATTCCAGCTGACCACCATGCCGCATTTAGCCGCCGAAG (SEQ ID NO: 622)YJL131C GGAATTCCAGCTGACCACCATGTTAAAAGTTCCTTTGAGTGATGT (SEQ ID NO: 623)YJL151C GGAATTCCAGCTGACCACCATGGACAGAGACCATATTAATGACC (SEQ ID NO: 624)YLR441C GGAATTCCAGCTGACCACCATGGCTGTCGGAAAGAATAAGAGA (SEQ ID NO: 625)YLR415C GGAATTCCAGCTGACCACCATGTATCTCAGTGCCCAGCTTATG (SEQ ID NO: 626)YLR212C GGAATTCCAGCTGACCACCATGGGTGGAGAAATTATTACTTTGC (SEQ ID NO: 627)YLR029C GGAATTCCAGCTGACCACCATGGGTGCCTACAAATATTTGGAAG (SEQ ID NO: 628)YLL041C GGAATTCCAGCTGACCACCATGTTGAACGTGCTATTGAGAAGGA (SEQ ID NO: 629)YLR105C GGAATTCCAGCTGACCACCATGTCTAAAGGGAGGGTCAATCAG (SEQ ID NO: 630)YIL136W GGAATTCCAGCTGACCACCATGTCATCAAGAATAATTGTCGGCA (SEQ ID NO: 631)YLR215C GGAATTCCAGCTGACCACCATGTCCTCACAAGAATATACAACTTT (SEQ ID NO: 632)YLR321C GGAATTCCAGCTGACCACCATGTCGCACCAAAACCAGCTTATT (SEQ ID NO: 633)YMR260C GGAATTCCAGCTGACCACCATGGGTAAGAAAAACACTAAAGGTG (SEQ ID NO: 634)YNL120C GGAATTCCAGCTGACCACCATGATAAAAGTCGATACTTCCGATG (SEQ ID NO: 635)YLR407W GGAATTCCAGCTGACCACCATGACTGTTTCTACTTCCAAGACC (SEQ ID NO: 636)YMR197C GGAATTCCAGCTGACCACCATGAGTTCCCTATTAATATCATACGA (SEQ ID NO: 637)YMR100W GGAATTCCAGCTGACCACCATGAGAGACTCTAATCATCGATCAT (SEQ ID NO: 638)YMR210W GGAATTCCAGCTGACCACCATGCGTCTAAAAGAATTGTTACCTAA (SEQ ID NO: 639)YMR318C GGAATTCCAGCTGACCACCATGTCTTATCCTGAGAAATTTGAAGG (SEQ ID NO: 640)YMR069W GGAATTCCAGCTGACCACCATGCGTTCTTCGGTATATAGTGAGA (SEQ ID NO: 641)YNL076W GGAATTCCAGCTGACCACCATGTCGCGGGAGGCATTTGATGT (SEQ ID NO: 642)YNL024C GGAATTCCAGCTGACCACCATGGAGAGTATATTTGGTGGGTTTG (SEQ ID NO: 643)YNL125C GGAATTCCAGCTGACCACCATGTCAACGCACTCAAACGACTAC (SEQ ID NO: 644)YNL029C GGAATTCCAGCTGACCACCATGTTGCTAATAAGAAGGACGATAAA (SEQ ID NO: 645)YMR115W GGAATTCCAGCTGACCACCATGCTTTTACAAGGAATGCGTTTATC (SEQ ID NO: 646)YNL244C GGAATTCCAGCTGACCACCATGTCCATTGAGAATCTGAAATCATT (SEQ ID NO: 647)YNL334C GGAATTCCAGCTGACCACCATGACCGTCGTTATCGGAGTCTT (SEQ ID NO: 648)YNR018W GGAATTCCAGCTGACCACCATGAAGATTTTAACCCAAGACGAAAT (SEQ ID NO: 649)YNL277W GGAATTCCAGCTGACCACCATGTCGCATACTTTAAAATCGAAAAC (SEQ ID NO: 650)YOL118C GGAATTCCAGCTGACCACCATGTCTTTTAGAAAGAAAAAACTCAAAC (SEQ ID NO: 651)YOL123W GGAATTCCAGCTGACCACCATGAGCTCTGACGAAGAAGATTTCA (SEQ ID NO: 652)YOR020C GGAATTCCAGCTGACCACCATGTCCACCCTTTTGAAGTCTGCT (SEQ ID NO: 653)YOL116W GGAATTCCAGCTGACCACCATGGCAAGTAACCAGCACATAGGA (SEQ ID NO: 654)YOR305w GGAATTCCAGCTGACCACCATGATAAAAAACTATTTGGGACGAAG (SEQ ID NO: 655)YPL267W GGAATTCCAGCTGACCACCATGATATCACCATCAAAAAAGAGAAC (SEQ ID NO: 656)YPL229w GGAATTCCAGCTGACCACCATGATGCCCTACAACACCCCTC (SEQ ID NO: 657)YPL038W GGAATTCCAGCTGACCACCATGAAACTGGCGCAAGACATGAAT (SEQ ID NO: 658)YPR047H GGAATTCCAGCTGACCACCATGGAGGTAACTTCAATGTTTCTCA (SEQ ID NO: 659)YPL011C GGAATTCCAGCTGACCACCATGACTACAAATAATGACTTCTATTTTG (SEQ ID NO: 660)YPR148C GGAATTCCAGCTGACCACCATGTCTGGTTATTTTTCAGGGTTTTC (SEQ ID NO: 661)YOL103W GGAATTCCAGCTGACCACCATGGCTGAAATGAAGAATTCGACAG (SEQ ID NO: 662)YOR016C GGAATTCCAGCTGACCACCATGCGCGTTTTTACTTTGATTGCGA (SEQ ID NO: 663)YPL079W GGAATTCCAGCTGACCACCATGGGTAAATCGTATGTCCATATAAC (SEQ ID NO: 664)YOR260W GGAATTCCAGCTGACCACCATGTCAATTCAGGCTTTTGTCTTTTG (SEQ ID NO: 665)YOR360C GGAATTCCAGCTGACCACCATGTCCACCCTTTTTCTGATTGGAA (SEQ ID NO: 666)YDL060W GGAATTCCAGCTGACCACCATGGCAGGTCATTCACACAGGTC (SEQ ID NO: 667)YDL005C GGAATTCCAGCTGACCACCATGGTAGTACAAAATAGCCCAGTTT (SEQ ID NO: 668)YPL210C GGAATTCCAGCTGACCACCATGAAAGAAAGCAAAAAAATGGCTAAA (SEQ ID NO: 669)YMR118C GGAATTCCAGCTGACCACCATGAAAGCAACCATTCAAAGAGTAAC (SEQ ID NO: 670)YPR052C GGAATTCCAGCTGACCACCATGGTCACCCCAAGAGAACCTAA (SEQ ID NO: 671)YLR224W GGAATTCCAGCTGACCACCATGAATCAGAGCGATAGCAGCTTG (SEQ ID NO: 672)YLR275W GGAATTCCAGCTGACCACCATGTCGTATGTTTGATCTTAACCATT (SEQ ID NO: 673)YMR154C GGAATTCCAGCTGACCACCATGAATGATTGGCATGAGTTCAATG (SEQ ID NO: 674)YDR205w GGAATTCCAGCTGACCACCATGGATAGAGGCAGGTGGTGTTT (SEQ ID NO: 675)YPR037C GGAATTCCAGCTGACCACCATGAAACAGATAGTCAAAAGAAGCC (SEQ ID NO: 676)YNR008W GGAATTCCAGCTGACCACCATGGGCACACTGTTTCGAAGAAAT (SEQ ID NO: 677)YOR084W GGAATTCCAGCTGACCACCATGGAACAGAACAGGTTCAAGAAAG (SEQ ID NO: 678)YGR054W GGAATTCCAGCTGACCACCATGTCATCTCAGTTTTTCCTGAAAAC (SEQ ID NO: 679)YGL106W GGAATTCCAGCTGACCACCATGTCAGCCACCAGAGCCAATAAA (SEQ ID NO: 680)YAL067C GGAATTCCAGCTGACCACCATGTATTCAATTGTTAAAGAGATTATTG (SEQ ID NO: 681)YIL023c GGAATTCCAGCTGACCACCATGAAGGCGTCGCACATTTGCTC (SEQ ID NO: 682)YBR064W GGAATTCCAGCTGACCACCATGGATATGGTATCACCAGTCTTGA (SEQ ID NO: 683)b0019 TTGCTCTTCCATGAAACATCTGCATCGATTCTTTAG (SEQ ID NO: 684) b2148TTGCTCTTCCATGAGTGCGTTAAATAAGAAAAG (SEQ ID NO: 685) b2796TTGCTCTTCCATGGAAACGACTCAAACCAGCAC (SEQ ID NO: 686) b2082TTGCTCTTCCATGTTTCATTGTCCTTTATGCCAGC (SEQ ID NO: 687) b0124TTGCTCTTCCATGGCAATTAACAATACAGGCTCG (SEQ ID NO: 688) b3116TTGCTCTTCCATGAGTACTTCAGATAGCATTGTATC (SEQ ID NO: 689) b1830TTGCTCTTCCATGAACATGTTTTTTAGGCTTACC (SEQ ID NO: 690) b1453TTGCTCTTCCATGTTCATGGCAACTTATATGACTTTT (SEQ ID NO: 691) b2664TTGCTCTTCCATGATCAGGAGTCACACCATGA (SEQ ID NO: 692) b2799TTGCTCTTCCATGATGGCTAACAGAATGATTCTGA (SEQ ID NO: 693) b3327TTGCTCTTCCATGAATTATCGCTATCGCGCCA (SEQ ID NO: 694) b0970TTGCTCTTCCATGGATCGTATTGTTAGTTCTTCAC (SEQ ID NO: 695) YER003CGGAATTCCAGCTGACCACCATGTCCAACAAGCTGTTCAGGTTA (SEQ ID NO: 696) YCL027WGGAATTCCAGCTGACCACCATGGTAGCAACAATAATGCAGACGA (SEQ ID NO: 697) YBR112CGGAATTCCAGCTGACCACCATGAATCCGGGCGGTGAACAAAC (SEQ ID NO: 698) YNL079CGGAATTCCAGCTGACCACCATGGACAAAATCAGAGAAAAGCTAAG (SEQ ID NO: 699) YFR042WGGAATTCCAGCTGACCACCATGGCAGGTATCAAGTTGACGCAT (SEQ ID NO: 700) YER137CGGAATTCCAGCTGACCACCATGTGTGAATCATCAAATAAGACTGA (SEQ ID NO: 701) YKL103CGGAATTCCAGCTGACCACCATGGAGGAACAACGTGAAATACTG (SEQ ID NO: 702) YNL090WGGAATTCCAGCTGACCACCATGTCTGAAAAGGCCGTTAGAAGG (SEQ ID NO: 703) YGR161CGGAATTCCAGCTGACCACCATGATCGCTACCTCCAGAGCCG (SEQ ID NO: 704) YDR071CGGAATTCCAGCTGACCACCATGGCCTCCTCAAGTAGCACGC (SEQ ID NO: 705) Reverse SeqYGL263W GATCCCCGGGAATTGCCATGTTACACATCATTGCAAGCTGATTGT (SEQ ID NO: 706)YGR004W GATCCCCGGGAATTGCCATGTTATAGAGAAGGAGACATTGAAACAT (SEQ ID NO: 707)YGR014W GATCCCCGGGAATTGCCATGTCAAACTTCGTTCCAACCCAGGG (SEQ ID NO: 708)YGL239C GATCCGCGGGAATTGCCATGTCAATTGCAGGGATTATGGAATAAAA (SEQ ID NO: 709)YBL060W GATCCCCGGGAATTGCCATGTTAGAACTGAACAGAACCCATGGC (SEQ ID NO: 710)YGL166W GATCCCCGGGAATTGCCATGTTATTGTGAATGTGAGTTATGCGAAG (SEQ ID NO: 711)YDL202W GATCCCCGGGAATTGCCATGTTACTTTGATCCCTTCGATTCTGCA (SEQ ID NO: 712)YAL046C GATCCCCGGGAATTGCCATGTCATGATGATGCCGGACCCTTC (SEQ ID NO: 713)YDR101C GATCCCCGGGAATTGCCATGCTACATTTTCATGGTTTCTTCAACTC (SEQ ID NO: 714)YDR108W GATCCCCGGGAATTGCCATGTCATCCAATAAAGCTAACACTTGTTC (SEQ ID NO: 715)YAL064W GATCCCCGGGAATTGCCATGCTATGGTTCGCTATTCAATATTAGAA (SEQ ID NO: 716)YDR134C GATCCCCGGGAATTGCCATGTTACAACAATAAAGCGGCAGCACC (SEQ ID NO: 717)YFL031w GATCCCCGGGAATTGCCATGTCAACAGCAGCCCCCACCGGT (SEQ ID NO: 718)YFL052W GATCCCCGGGAATTGCCATGTTAAGGAAGCGCATCTACATCTTCT (SEQ ID NO: 719)YFL042C GATCCCCGGGAATTGCCATGTCAACCATACCTTTGATCCAACTG (SEQ ID NO: 720)YBR025C GATCCCCGGGAATTGCCATGTCAATTCTTACCAGCACCAGCTCT (SEQ ID NO: 721)YER174C GATCCCCGGGAATTGCCATGTTACTGTAGAGCATGTTGGAAATATT (SEQ ID NO: 722)YBR051W GATCCCCGGGAATTGCCATGTTATATATGGCATGTCTTCGCATGT (SEQ ID NO: 723)YER175C GATCCCCGGGAATTGCCATGTCAGACCCTTTTGCCAAGTTTGTAA (SEQ ID NO: 724)YDR521W GATCCCCGGGAATTGCCATGTTACTCACCATTAAAACATCTTTCCC (SEQ ID NO: 725)YER167WGATCCCCGGGAATTGCCATGTTAGTTGCTATTATCAAAATAAAAAGAC (SEQ ID NO: 726)YER123W GATCCCCGGGAATTGCCATGTCAAAAAAAAAAAGGAAAAAGAGAAAAG (SEQ ID NO:727)YDR415C GATCCCCGGGAATTGCCATGTCACATTTTTCTAAATTCACTTAGCAC (SEQ ID NO: 728)YEL052W GATCCCCGGGAATTGCCATGTTAGTATGTAGGCTTAGTAACCCAA (SEQ ID NO: 729)YDR536W GATCCCCGGGAATTGCCATGTCAACCCTCAAAATTTGCTTTATCG (SEQ ID NO: 730)YDR513W GATCCCCGGGAATTGCCATGCTATTGAAATACCGGCTTCAATATTT (SEQ ID NO: 731)YEL045C GATCCCCGGGAATTGCCATGCTAGGAAAGGAGGTGGTTACGAA (SEQ ID NO: 732)YEL041W GATCCCCGGGAATTGCCATGTTAGATTGCAAAATGAGCCTGACGA (SEQ ID NO: 733)YDL238C GATCCCCGGGAATTGCCATGCTAAATCTGGTAGACTTGCTGGC (SEQ ID NO: 734)YBR282W GATCCCCGGGAATTGCCATGTTATCCACGCTCCTTATAACATGAA (SEQ ID NO: 735)YBR258C GATCCCCGGGAATTGCCATGTTACGTACTTCCATTTGCTTCCTCT (SEQ ID NO: 736)YCL001W-AGATCCCCGGGAATTGCCATGTCAGTTCATCAAAATTGAAATTTCTAACCA (SEQ ID NO: 737)YBR274W GATCCCCGGGAATTGCCATGTCAGTTGGGAATTAGGATAATATCC (SEQ ID NO: 738)YHR090C GATCCCCGGGAATTGCCATGTCAGTTACGTTTTCTTTTCAGTTTGT (SEQ ID NO: 739)YGR121C GATCCCCGGGAATTGCCATGTTACCTATTGGCAGGATCTTCTTGA (SEQ ID NO: 740)YGR127WGATCCCCGGGAATTGCCATGTTACAATTTGAATTTAAACCTTTTTTCC (SEQ ID NO: 741)YGR150C GATCCCCGGGAATTGCCATGCTACATGTTAAGTTCTTGTTCCTCC (SEQ ID NO: 742)YKL037W GATCCCCGGGAATTGCCATGTTATATACTCAATCCAAAACAGGGAA (SEQ ID NO: 743)YKL051W GATCCCCGGGAATTGCCATGTCATACGACTACTTGAATAGATTCG (SEQ ID NO: 744)YKL120W GATCCCCGGGAATTGCCATGTTAATTATGGCCTAAAACTCTCGAC (SEQ ID NO: 745)YKL011C GATCCCCGGGAATTGCCATGTTAGTCATTGTTGTAAGTGTTCTGC (SEQ ID NO: 746)YKL017C GATCCCCGGGAATTGCCATGTTACAAATAATCGTCAATGTTGGGG (SEQ ID NO: 747)YKL049C GATCCCCGGGAATTGCCATGCTAAATAAACTGTCCCCTGATTCTT (SEQ ID NO: 748)YKL132C GATCCCCGGGAATTGCCATGCTATACTGGCAAGTGACAGTTGTG (SEQ ID NO: 749)YGR126W GATCCCCGGGAATTGCCATGTTAATCGAAAATTCTATGAAAAAACCC (SEQ ID NO: 750)YKL070W GATCCCCGGGAATTGCCATGTCAGAAACGCTCCACTTTACTTCG (SEQ ID NO: 751)YKL058W GATCCCCGGGAATTGCCATGTTACTCGCTCTTTTTTGAGTTACATG (SEQ ID NO: 752)YHR130C GATCCCCGGGAATTGCCATGCTAATTCCTGATGCCAAGTAACGA (SEQ ID NO: 753)YIL070C GATCCCCGGGAATTGCCATGTTAGTGGAAAAACTTCTTCATCTTTTC (SEQ ID NO: 754)YHR195W GATCCCCGGGAATTGCCATGTTAGTATCTAAATGGTTGAGAGTATG (SEQ ID NO: 755)YIR022W GATCCCCGGGAATTGCCATGCTACTCGCCCCCCAGCAGAG (SEQ ID NO: 756)YJL089W GATCCCCGGGAATTGCCATGTTAGAAGGTCGAGTTCAAAATATTCT (SEQ ID NO: 757)YJL172W GATCCCCGGGAATTGCCATGTTAAGCGTATTCGTTAACATTAACGA (SEQ ID NO: 758)YHR113W GATCCCCGGGAATTGCCATGTTAGACAACAATTTCAGATTCTATGG (SEQ ID NO: 759)YHR175W GATCCCCGGGAATTGCCATGTTAATGGCAGGCGAGGGAGCTG (SEQ ID NO: 760)YGR212WGATCCCCGGGAATTGCCATGCTAGTATAAATTTAAGTAATCTTTCATAT (SEQ ID NO: 761)YJL024C GATCCCCGGGAATTGCCATGTTATTGCCCCGTTGCCCATTGTG (SEQ ID NO: 762)YGR180c GATCCCCGGGAATTGCCATGTTAGAAGTCATCATCAAAGTTAATTTC (SEQ ID NO: 763)YJL179W GATCCCCGGGAATTGCCATGTTAATTCTTCATCAATGCCTTTAGATT (SEQ ID NO: 764)YJL001W GATCCCCGGGAATTGCCATGTTATAGTTGTTCATATTCATCAGGGT (SEQ ID NO: 765)YJL208C GATCCCCGGGAATTGCCATGTCAATTCCTTTTTTTTGGAGGAGGT (SEQ ID NO: 766)YJL152W GATCCCCGGGAATTGCCATGTCACGCAGACATGCGACTGCG (SEQ ID NO: 767)YJL131CGATCCCCGGGAATTGCCATGTTACATTTCATTCATTTTTTTTCTCTGA (SEQ ID NO: 768)YJL151C GATCCCCGGGAATTGCCATGCTAAGTACGGCCGGAAGAGAGC (SEQ ID NO: 769)YLR441C GATCCCCGGGAATTGCCATGTTACACAGTTTCCAAGACTTCGTC (SEQ ID NO: 770)YLR415C GATCCCCGGGAATTGCCATGCTACTTCCAAACAACTGGTCCAGA (SEQ ID NO: 771)YLR212C GATCCCCGGGAATTGCCATGTTATACTAATTTATGATCACCGTCGG (SEQ ID NO: 772)YLR029C GATCCCCGGGAATTGCCATGTTATTTTCTGTATCTCCACAAGGAC (SEQ ID NO: 773)YLL041C GATCCCCGGGAATTGCCATGCTAGGCAAATGCCAAAGATTTCTTA (SEQ ID NO: 774)YLR105C GATCCCCGGGAATTGCCATGCTAGTCTCTATTTCTTCCGGGAAC (SEQ ID NO: 775)YIL136W GATCCCCGGGAATTGCCATGCTAGTCCTTTTTCGAGCTCCAGAA (SEQ ID NO: 776)YLR215C GATCCCCGGGAATTGCCATGCTAAGTTTCATTCTCACTATCACTG (SEQ ID NO: 777)YLR321C GATCCCCGGGAATTGCCATGCTACATTCTCATTGTGGTTTCTAAG (SEQ ID NO: 778)YMR260CGATCCCCGGGAATTGCCATGTTAATCATAAATAGTTTCATAAGTGTGT (SEQ ID NO: 779)YNL120C GATCCCCGGGAATTGCCATGTCACTTCCTATGCAAAATGCTTAATA (SEQ ID NO: 780)YLR407W GATCCCCGGGAATTGCCATGTCAGTCATGGCATGCCTTGGCA (SEQ ID NO: 781)YMR197C GATCCCCGGGAATTGCCATGCTAAAATGAAGACAGCCACAATCTG (SEQ ID NO: 782)YMR100W GATCCCCGGGAATTGCCATGCTATTGATTGTTTGTTCCACGGACT (SEQ ID NO: 783)YMR210W GATCCCCGGGAATTGCCATGTTATGAAGTCCATGGTAAATTCGTG (SEQ ID NO: 784)YMR318C GATCCCCGGGAATTGCCATGTTACATGAGGTTCATGTTCATGTTAG (SEQ ID NO: 785)YMR069WGATCCGCGGGAATTGCCATGTTATTTTTTACTTAATTTCATCCATTTAG (SEQ ID NO: 786)YNL076WGATCCCCGGGAATTGCCATGTTATGTATCACAACTATTAAATTCAGTT (SEQ ID NO: 787)YNL024C GATCCCCGGGAATTGCCATGTTAATCTCTTTCAAAACAGACAGCAA (SEQ ID NO: 788)YNL125C GATCCCCGGGAATTGCCATGCTATTTTTCACTTTGGCTGTTGCC (SEQ ID NO: 789)YNL029C GATCCCCGGGAATTGCCATGTTAACTCTCTTGTCCCGATTTCTC (SEQ ID NO: 790)YMR115W GATCCCCGGGAATTGCCATGTTAGTTTCTCAAGTGACTATTGTGAA (SEQ ID NO: 791)YNL244C GATCCCCGGGAATTGCCATGTTACGAATCAGTCCGATTGGACTT (SEQ ID NO: 792)YNL334C GATCCCCGGGAATTGCCATGTTAAGCTGGAAGAGCCAATCTCTT (SEQ ID NO: 793)YNR018WGATCCCCGGGAATTGCCATGTTATTTCAAAGTCTTCAACAATTTTTCT (SEQ ID NO: 794)YNL277W GATCCCCGGGAATTGCCATGTTAGTCTTCATGCTTATCACAGAAC (SEQ ID NO: 795)YOL118C GATCCCCGGGAATTGCCATGTCACTTCAAAGTCTCTGGAATATGA (SEQ ID NO: 796)YOL123W GATCCCCGGGAATTGCCATGTTATTCGTCTTCTTCCAAAGTTTGAG (SEQ ID NO: 797)YOR020C GATCCCCGGGAATTGCCATGCTATGAGCGATCCCGTTTTGTGAA (SEQ ID NO: 798)YOL116WGATCCCCGGGAATTGCCATGTTAATAATTGAATTTAATTTTACTTCTGTT (SEQ ID NO: 799)YOR305w GATCCCCGGGAATTGCCATGTCACCGACTCATTTTGTTAAGCTTG (SEQ ID NO: 800)YPL267W GATCCCCGGGAATTGCCATGTTACTCATCTTCATAGACGTGGAAG (SEQ ID NO: 801)YPL229wGATCCCCGGGAATTGCCATGCTAACATTTCTTATTATCTCTATATATC (SEQ ID NO: 802)YPL038W GATCCCCGGGAATTGCCATGCTAACTGCTTTTTTCGTGTTGAGTA (SEQ ID NO: 803)YPR047WGATCCCCGGGAATTGCCATGTCATTTTTGTCCCTTTATATCAATTTTT (SEQ ID NO: 804)YPL011C GATCCCCGGGAATTGCCATGTTAGCGTTTTTTTTTGCCCTTCTTC (SEQ ID NO: 805)YPR148C GATCCCCGGGAATTGCCATGCTAAATTTCCAAACCATTTAGAACTTT (SEQ ID NO: 806)YOL103W GATCCCCGGGAATTGCCATGCTAAGACTTGAAATTAATTAATTCGGG (SEQ ID NO: 807)YOR016C GATCCCCGGGAATTGCCATGTCACTGTATCTCGCTGTCACAATC (SEQ ID NO: 808)YPL079W GATCCCCGGGAATTGCCATGCTATTGTAATTCCCTTATAGTGTTCA (SEQ ID NO: 809)YOR260W GATCCCCGGGAATTGCCATGTTATTTAAGCTCGAAATGGCTATTGA (SEQ ID NO: 810)

Vector Preparation

The preferred binary vector 1bxbigResgen for yeast and 1bxSuperCoLic forE. coli, which is based on the modified pPZP binary vector backbone(comprising the kanamycin-gene for bacterial selection; Hajukiewicz, P.et al., 1994, Plant Mol. Biol., 25: 989-994) carried the selectionmarker bar-gene (De Block et al., 1987, EMBO J. 6, 2513-2518) driven bythe mas1′ promotor (Velten et al., 1984, EMBO J. 3, 2723-2730; Mengiste,Amedeo and Paszkowski, 1997, Plant J., 12, 945-948) on its T-DNA.

In addition the T-DNA contained the strong double 35S (Kay et al., 1987,Science 236, 1299-1302) for yeast or Super promotor (Ni et al., 1995,Plant Journal 7, 661-676) for E. coli in front of a cloning cassettefollowed by the nos-terminator (Depicker A. Stachel S. Dhaese P.Zambryski P. Goodman H M. Nopaline synthase: transcript mapping and DNAsequence. Journal of Molecular & Applied Genetics. 1(6):561-73, 1982.).The cloning cassette consists of the following sequence:

′Yeast: 5′-GGAATTCCAGCTGACCACCATGGCAATTCCCGGGGATC-3′ (SEQ ID NO: 851) or

E. coli: 5′-TTG CTC TTC CAT GGC AAT GAT TAA TTA ACG AAG AGC AA-3′ (SEQID NO: 852), respectively.

Other selection marker systems, like the AHAS marker or other promotors,e.g. superpromotor (see above), 35S promotor (see above), Ubiquitinpromotor (Callis et al., J. Biol. Chem., 1990, 265: 12486-12493; U.S.Pat. No. 5,510,474; U.S. Pat. No. 6,020,190; Kawalleck et al., Plant.Molecular Biology, 1993, 21: 673-684) or 34S promotor (GenBank Accessionnumbers M59930 and X16673) were similar useful for the instant inventionand are known to a person skilled in the art. The vector was linearisedwith EcoR and SmaI for yeast or SmaI and SacI for E. coli using thestandard protocol provided by the supplier (MBI Fermentas, Germany) andpurified using Qiagen columns (Qiagen, Hilden, Germany).

Ligation and Transformation

Present ORF fragments (˜100 ng) were digested by EcoRI and SmaI foryeast and SmaI and SacI for E. coli using the standard protocol providedby the supplier (MBI Fermentas, Germany), purified using Qiagen columns(Qiagen, Hilden, Germany) and were ligated into the cloning cassette ofthe binary vector systems (˜30 ng) using standard procedures (Maniatiset al.).

In the case of internal EcoRI, SmaI and SacI restriction sites a bluntend cloning procedure was applied. The undigested ORF fragments weredirectly purified and ligated into the cloning cassette of the binaryvector. In this case the EcoRI site was refilled by Klenow reaction andthe SacI site blunted Pfu DNA polymerase.

Ligation products were transformed into E. coli (DH5alpha) using astandard heat shock protocol (Maniatis et al.). Transformed colonieswere grown on LB media and selected by respective antibiotica (Km) for16 h at 37° C. Positive clones were identified by control PCR reactionsusing a combination of a vector specific and the respective ORF specificprimers.

Plasmid Preparation

Plasmid DNA was prepared from positive clones using standard protocols(Qiagen Hilden, Germany).

Transformation of Agrobacteria

Plasmids were transformed into Agrobacterium tumefaciens (GV3101 pMP90;Koncz and Schell, 1986, Mol. Gen. Genet. 204: 383-396) using heat shockor electroporation protocols. Transformed colonies were grown on YEPmedia and selected by respective antibiotics (Rif/Gent/Km) for 2 d at28° C. These Agrobacterium cultures were used for the planttransformation.

Arabidopsis thaliana was grown and transformed according to standardconditions Bechtold 1993 (Bechtold, N., Ellis, J., Pelletier, G. 1993.In planta Agrobacterium mediated gene transfer by infiltration ofArabidopsis thaliana plants C. R. Acad. Sci. Paris. 316:1194-1199); Bentet al. 1994 (Bent, A., Kunkel, B. N., Dahlbeck, D., Brown, K. L.,Schmidt, R., Giraudat, J., Leung, J., and Staskawicz, B. J. 1994; PPCS2of Arabidopsis thaliana: A leucin-rich repeat class of plant diseaseresistant genes; Science 265: 1856-1860).

Transgenic A. thaliana plants were grown individually in pots containinga 4:1 (v/v) mixture of soil and quartz sand in a York growth chamber.Standard growth conditions were: photoperiod of 16 h light and 8 h dark,20° C., 60% relative humidity, and a photon flux density of 150 μE. Toinduce germination, sown seeds were kept at 4° C., in the dark, for 3days. Plants were watered daily until they were approximately 3 weeksold at which time drought was imposed by withholding water. Parallely,the relative humidity was reduced in 10% increments every second day to20%. After approximately 12 days of withholding water, most plantsshowed visual symptoms of injury, such as wilting and leaf browning,whereas tolerant plants were identified as being visually turgid andhealthy green in color. Plants were scored for symptoms of droughtinjury in comparison to neighbouring plants for 3 days in succession.

Three successive experiments were conducted. In the first experiment,independent T2 lines were sown for each gene being tested. Thepercentage of plants not showing visual symptoms of injury wasdetermined. In the second experiment, the lines that had been scored astolerant in the first experiment were put through a confirmation screenaccording to the same experimental procedures. In this experiment,plants of each tolerant line were grown and treated as before. In thethird experiment, at least 7 replicates of the most tolerant line weregrown and treated as before. The average and maximum number of days ofdrought survival after wild-type control had visually died weredetermined. Additionally measurements of chlorophyll fluorescence weremade in stressed and non-stressed plants using a Mini-PAM (Heinz WalzGmbH, Effeltrich, Germany).

In the first experiment, after 12 days of drought, the control,non-transgenic Arabidopsis thaliana and most transgenic lines expressingother transgenes in the test showed extreme visual symptoms of stressincluding necrosis and cell death. Several plants expressing the genesretained viability as shown by their turgid appearance and maintenanceof green color.

The second experiment compared a smaller number of independenttransgenic lines for each gene but a greater number of progeny withineach independent transformation event. This experiment confirmed theprevious results. Those lines containing the specific SRP encoding yeastgenes survived longer than the controls. In some cases the transgenicline survived more than 3 days after the controls had died.

According to the results of the first and second experiments some majorlines containing the specific SRP encoding yeast genes were identified,which showed the best results with regard to the average days ofsurvival after wild type and/or the hit percentage.

In a third experiment these major lines were tested with multiplereplicates (4-80 plants per line). The average number of days the plantsof the major line survived longer than wild-type was measured. I.e., thenumber ‘1’ means that, on average, the plants overexpressing this ORF,on averaged survived 1 day longer than wild-type. The value for WT inthis column is ‘0’. The results are summmarised in table 3.

TABLE 3 Drought tolerance of transgenic Arabidopsis thaliana expressingthe various SRP encoding genes from Saccharomyces cerevisiae or E. coliafter imposition of drought stress on 3 week old plants in a thirdexperiment using several plants from one transgenic line (experiment 3).Drought tolerance is measured for the indicated number of transgenicplants (Plants tested) as the average number of days (Average days ofsurvival after WT) that the transgenic plants survived after the control(untransformed wild type). The hit percentage indicates the fraction ofthe tested plants of the major line that was actually resistant, i.e.the number ‘50’ indicates that half of the tested plants were resistant(survived longer than WT). is column has the value ‘0’. Sequ. ID PlantsAverage days of survival No. Gene tested after WT 1 YGL263W 12 1.17 3YGR004W 11 1.36 5 YGR014W 11 0.82 7 YGL239C 13 2.4 9 YBL060W 14 1.6 11YGL166W 14 1.07 13 YDL202W 15 0.47 15 YAL046C 14 1.9 17 YDR101C 14 3.5719 YDR108W 14 0.5 21 YAL064W 33 2.29 23 YDR134C 13 0.8 26 YFL031w 13 1.328 YFL052W 14 1.1 30 YFL042C 11 1.3 32 YBR025C 11 1.4 34 YER174C 22 1.0536 YBR051W 10 1.2 38 YER175C 11 1.7 40 YDR521W 14 0.5 42 YER167W 35 0.6644 YER123W 11 2.2 46 YDR415C 7 2.71 48 YEL052W 4 1.5 50 YDR536W 14 1.552 YDR513W 7 3.14 54 YEL045C 14 1.64 56 YEL041W 13 1.77 58 YDR415C 72.71 60 YDL238C 35 1.2 62 YBR282W 14 1.79 64 YBR258C 9 3.4 66 YCL001W-A36 1.78 68 YBR274W 14 2 70 YHR090C 8 4.5 72 YGR121C 40 1.6 74 YGR127W 81.4 76 YGR150C 12 2.6 78 YKL037W 14 0.79 80 YKL051W 16 2.4 82 YKL120W 140.64 84 YKL011C 12 1.9 86 YKL017C 10 1.8 88 YKL049C 80 1.92 90 YKL132C33 1.82 92 YGR126W 8 2.3 94 YKL070W 14 2.1 96 YKL058W 9 1.44 98 YHR130C9 1.8 100 YIL070C 13 0.69 102 YHR195W 14 1.9 104 YIR022W 14 1.07 106YJL089W 14 0.86 108 YJL172W 11 0.82 110 YHR113W 15 2 112 YHR175W 9 0.78114 YJL024C 13 0.69 116 YGR180c 14 2.9 118 YJL179W 18 1.3 120 YJL001W 141.6 122 YJL208C 12 1.4 124 YJL152W 13 1.3 126 YJL131C 14 0.6 128 YJL151C14 1.9 130 YLR441C 10 2 132 YLR415C 14 1.6 134 YLR212C 13 0.64 136YLR029C 14 1.56 137 YLL041C 13 0.92 139 YLR105C 14 0.86 141 YIL136W 82.25 143 YLR215C 13 1.77 145 YLR321C 14 1.29 147 YMR260C 11 2.09 149YNL120C 7 2 151 YLR407W 12 1.17 153 YMR197C 14 0.57 155 YMR100W 12 1.25157 YMR210W 10 1.1 159 YMR318C 13 0.85 161 YMR069W 8 1.25 163 YNL076W 131.31 165 YNL024C 13 1.08 167 YNL125C 4 1.75 169 YNL029C 13 1.92 171YMR115W 12 0.75 173 YNL244C 11 1.55 175 YNL334C 14 1.5 177 YNR018W 141.29 179 YNL277W 14 1.14 181 YOL118C 14 1.71 183 YOL123W 14 0.71 185YOR020C 12 1.83 187 YOL116W 13 1.08 189 YOR305w 15 1.2 191 YPL267W 6 2.5193 YPL229w 5 2 195 YPL038W 10 1.3 197 YPR047W 11 1 199 YPL011C 12 0.75201 YPR148C 10 1.1 203 YOL103W 12 0.75 205 YOR016C 14 0.79 207 YPL079W15 1.33 209 YOR260W 7 1.29 211 YOR360C 15 1.53 213 YDL060W 15 0.67 215YDL005C 15 1 217 YPL210C 15 1.13 219 YMR118C 14 1.14 221 YPR052C 14 1.07223 YLR224W 10 2.1 225 YLR275W 9 2.44 227 YMR154C 15 1.27 229 YDR205w 121.08 231 YPR037C 12 2.17 233 YNR008W 14 2.29 235 YOR084W 10 2.2 237YGR054W 14 1.5 239 YGL106W 13 3.46 241 YAL067C 13 1.62 243 YIL023c 151.73 245 YBR064W 15 1.13 247 b0020 13 0.78 249 b2148 15 3.13 251 b279615 2.33 253 b2082 14 2.43 255 b0124 15 2.87 257 b3116 15 1.07 259 b183015 2.07 261 b1453 14 2.29 263 b2664 13 1.85 265 b2799 15 1.87 267 b332715 1.47 269 b0970 15 1.33 271 YER003C 5 1 273 YCL027W 9 0.56 275 YBR112C10 0.5 277 YNL079C 9 0.67 279 YFR042W 9 0.78 281 YER137C 3 0 283 YKL103C9 1 285 YNL090W 6 0.83 287 YGR161C 7 0.86 289 YDR071C 9 0.78

In a further experiment, for individual major lines, other linescontaining the same gene construct, but resulting from a differenttransformation event were tested. In these lines, the specific SRPencoding yeast genes is incorporated at a different site in the plantgenome. The results are summmarised in table 4 in accordance to table 3.The results demonstrate the dependence of the stress tolerance and/orresistance in plants on the expression of the SRP, rather than theinsertion event.

TABLE 4 Drought tolerance of transgenic Arabidopsis thaliana expressingselected SRP encoding genes from Saccharomyces cerevisiae or E. coliafter imposition of drought stress on 3 week old plants in a thirdexperiment using one plant from several independent transgenic lineseach (experiment 3). Drought tolerance is measured for the indicatednumber of transgenic plants (Plants tested) as the average number ofdays (Average days of survival after WT) that the transgenic plantssurvived after the control (untransformed wild type). The hit percentageindicates the fraction of the tested plants of the major line that wasactually resistant, i.e. the number ‘50’ indicates that half of thetested plants were resistant (survived longer than WT). For WT, thiscolumn has the value ‘0’. Number Sequ. ID other lines Average days ofNo. Gene tested survival after WT 1 YGL263W 7 1.43 3 YGR004W 8 1 5YGR014W 8 0.75 7 YGL239C 5 1 9 YBL060W 8 2 11 YGL166W 8 0.63 13 YDL202W8 0.25 15 YAL046C 7 1.3 17 YDR101C 9 1.1 19 YDR108W 9 0.22 21 YAL064W 83 23 YDR134C 6 2.2 26 YFL031w 9 2.3 28 YFL052W 5 1.4 32 YBR025C 5 1.2 34YER174C 9 0.5 36 YBR051W 6 1.3 38 YER175C 4 1.4 40 YDR521W 3 0.7 44YER123W 6 0.3 46 YDR415C 7 1.4 48 YEL052W 3 1.33 50 YDR536W 8 1.25 52YDR513W 4 1.5 54 YEL045C 5 1.2 56 YEL041W 8 0.88 60 YDL238C 6 0.17 62YBR282W 9 2.2 64 YBR258C 7 1.7 66 YCL001W-A 7 0.57 68 YBR274W 9 0.78 70YHR090C 6 2.7 72 YGR121C 9 0.8 74 YGR127W 6 2.5 76 YGR150C 5 3 78YKL037W 9 0.78 80 YKL051W 5 1.8 82 YKL120W 8 0.63 84 YKL011C 5 1.4 86YKL017C 5 0.6 88 YKL049C 9 1.4 90 YKL132C 7 0.7 92 YGR126W 6 1.3 94YKL070W 6 2 96 YKL058W 8 0.88 98 YHR130C 9 2.1 100 YIL070C 7 0.71 102YHR195W 9 2.1 104 YIR022W 9 1.22 106 YJL089W 7 0.6 108 YJL172W 4 1 110YHR113W 7 1.6 112 YHR175W 3 1 114 YJL024C 6 1.33 116 YGR180c 8 2.7 118YJL179W 8 1.8 120 YJL001W 9 0.7 122 YJL208C 6 1.7 124 YJL152W 8 0.3 126YJL131C 6 1 128 YJL151C 8 1.6 130 YLR441C 7 2.6 132 YLR415C 9 0.3 134YLR212C 7 2.14 136 YLR029C 8 0.25 137 YLL041C 7 0.86 139 YLR105C 7 0.29141 YIL136W 9 1.75 143 YLR215C 8 1.25 145 YLR321C 7 0.86 147 YMR260C 80.88 149 YNL120C 9 1.56 151 YLR407W 5 0.4 153 YMR197C 9 1.22 155 YMR100W8 0.88 157 YMR210W 8 0.88 159 YMR318C 8 0.63 161 YMR069W 9 0.44 163YNL076W 4 1.75 165 YNL024C 9 1.78 167 YNL125C 7 2.14 169 YNL029C 9 1.88171 YMR115W 9 1.44 173 YNL244C 8 0.25 175 YNL334C 9 1.33 177 YNR018W 91.22 179 YNL277W 8 1 181 YOL118C 9 0.89 183 YOL123W 8 0.88 185 YOR020C 90.44 187 YOL116W 9 1.67 189 YOR305w 7 0.28 191 YPL267W 4 0.75 193YPL229w 5 1.6 195 YPL038W 5 0.4 197 YPR047W 2 1 199 YPL011C 6 0.5 201YPR148C 9 0.33 203 YOL103W 9 0.33 205 YOR016C 9 0.56 211 YOR360C 8 0.38213 YDL060W 8 0.5 215 YDL005C 9 0.44 217 YPL210C 8 1.5 219 YMR118C 101.1 221 YPR052C 7 0.86 223 YLR224W 9 1.22 225 YLR275W 8 1.75 227 YMR154C9 1.11 229 YDR205w 4 1 231 YPR037C 5 3.4 233 YNR008W 8 0.75 235 YOR084W6 0.5 239 YGL106W 7 2.14 241 YAL067C 6 1.83 243 YIL023c 1 3 245 YBR064W7 0.71 247 b0020 4 1.5 249 b2148 10 0.1 251 b2796 11 0.72 253 b2082 91.22 255 b0124 9 3.3 257 b3116 8 1 259 b1830 7 1.71 261 b1453 8 1.13 263b2664 9 1 267 b3327 10 0.8 269 b0970 8 1.5 271 YER003C 12 2.08 273YCL027W 14 2.14 275 YBR112C 11 3.3 277 YNL079C 13 2.69 279 YFR042W 7 2.3281 YER137C 13 2.2 283 YKL103C 10 2.8 285 YNL090W 12 4.33 287 YGR161C 122.7 289 YDR071C 11 3

Chlorophyll fluorescence measurements of photosynthetic yield confirmedthat severe drought stress completely inhibited photosynthesis in thecontrol plants, but the transgenic major lines maintained photosyntheticfunction longer (Table 5).

TABLE 5 Drought tolerance of transgenic Arabidopsis thaliana expressingthe various SRP encoding genes from Saccharomyces cerevisiae or E. coliafter imposition of drought stress on 3 week old plants in a thirdexperiment using several plants from one transgenic line (experiment 3).Drought tolerance is reported as photosynthetic yield measured bychlorophyll fluorescence measured at three different time point duringthe drought stress experiment, and compared to the untransformed wildtype control. For each transgenic line, the average of 5 replicateplants is indicated, the wild type value is the average of 20-25 plantsmeasured in the same experiment. Photosynthetic PhotosyntheticPhotosynthetic yield yield yield 6 days after 10 days 14 days finalafter final after final Seq. ID No. Gene watering wild type wateringwild type watering wild type 1 YGL263W 751 766 765 654 264 106 3 YGR004W759 766 755 654 246 106 5 YGR014W 759 766 752 654 7 YGL239C 782 757 786610 9 YBL060W 743 757 782 610 108 16 11 YGL166W 752 736 747 709 13YDL202W 790 766 508 548 15 YAL046C 788 760 756 549 216 20 19 YDR108W 756736 739 709 0 20 23 YDR134C 757 760 765 549 273 20 26 YFL031w 763 760766 549 784 20 28 YFL052W 757 757 753 610 30 YFL042C 743 757 780 610 32YBR025C 763 760 762 549 631 20 36 YBR051W 741 760 696 549 456 20 38YER175C 749 757 627 610 140 16 44 YER123W 767 757 780 610 147 16 48YEL052W 750 736 773 710 177 20 50 YDR536W 753 736 772 709 293 20 52YDR513W 782 794 660 413 411 54 54 YEL045C 755 736 553 709 147 20 56YEL041W 758 736 769 709 129 20 62 YBR282W 759 760 724 549 221 20 64YBR258C 759 757 772 610 144 16 68 YBR274W 749 736 769 709 146 20 70YHR090C 749 760 765 549 620 20 74 YGR127W 740 549 576 20 76 YGR150C 771760 742 549 618 20 78 YKL037W 761 736 760 709 134 20 80 YKL051W 733 760740 549 153 20 82 YKL120W 759 736 518 709 84 YKL011C 750 760 694 549 43420 86 YKL017C 744 549 734 549 754 20 92 YGR126W 784 760 750 549 159 2094 YKL070W 774 760 734 549 244 20 96 YKL058W 752 766 765 654 495 20 98YHR130C 772 760 756 549 147 20 100 YIL070C 768 766 755 654 102 YHR195W753 757 693 610 141 16 104 YIR022W 761 736 771 709 108 YJL172W 756 766758 654 293 20 110 YHR113W 749 760 754 549 142 20 112 YHR175W 768 766758 654 465 106 114 YJL024C 762 766 758 654 736 106 116 YGR180c 763 757779 610 118 YJL179W 744 760 606 549 310 20 120 YJL001W 748 757 519 610135 16 122 YJL208C 685 549 49 20 124 YJL152W 754 757 726 610 126 YJL131C750 757 758 610 128 YJL151C 755 760 764 549 152 20 130 YLR441C 745 760762 549 277 20 132 YLR415C 739 757 503 610 144 16 134 YLR212C 740 736759 709 103 20 136 YLR029C 746 736 780 709 292 20 139 YLR105C 751 736764 709 141 YIL136W 749 736 779 710 422 20 143 YLR215C 752 736 774 709151 20 145 YLR321C 749 736 767 709 145 20 147 YMR260C 774 766 763 654691 106 149 YNL120C 764 766 740 654 298 20 151 YLR407W 774 766 640 654138 106 153 YMR197C 751 736 723 709 63 20 155 YMR100W 770 766 596 654171 20 157 YMR210W 753 766 755 654 733 20 159 YMR318C 761 736 780 709135 20 161 YMR069W 756 766 750 654 519 20 163 YNL076W 765 766 757 654244 20 165 YNL024C 767 766 761 654 279 20 167 YNL125C 761 766 750 654283 20 169 YNL029C 764 766 758 654 171 YMR115W 755 736 739 709 173YNL244C 774 766 696 654 94 20 175 YNL334C 751 736 756 709 177 YNR018W756 736 749 709 181 YOL118C 727 736 756 709 280 20 183 YOL123W 747 736140 20 185 YOR020C 764 766 748 654 207 20 187 YOL116W 774 766 735 654135 20 189 YOR305w 773 769 565 245 191 YPL267W 757 767 756 548 193YPL229w 781 769 752 245 197 YPR047W 761 769 597 245 199 YPL011C 766 769582 245 201 YPR148C 771 769 401 245 203 YOL103W 789 769 237 245 205YOR016C 770 769 523 245 211 YOR360C 771 769 651 245 215 YDL005C 782 769702 245 217 YPL210C 794 769 735 245 219 YMR118C 777 768 499 272 221YPR052C 772 768 298 272 223 YLR224W 767 768 434 272 225 YLR275W 741 768780 272 227 YMR154C 760 768 734 272 229 YDR205w 787 768 241 272 231YPR037C 759 768 740 272 233 YNR008W 746 768 782 272 235 YOR084W 758 768765 272 237 YGR054W 766 768 140 272 239 YGL106W 760 768 477 272 241YAL067C 759 768 681 272 243 YIL023c 769 814 245 YBR064W 745 750 770 576117 31 249 b2148 736 768 740 272 251 b2796 761 768 319 272 253 b2082 756768 706 272 255 b0124 756 768 571 272 257 b3116 765 768 600 272 259b1830 757 768 772 272 261 b1453 750 768 648 272 263 b2664 764 768 521272 265 b2799 763 768 615 272 267 b3327 766 768 499 272 269 b0970 764768 560 272 271 YER003C 758 760 769 549 729 20 273 YCL027W 749 760 770549 145 20 275 YBR112C 760 760 760 549 731 20 277 YNL079C 763 766 762654 216 20 279 YFR042W 789 760 739 549 232 20 281 YER137C 760 760 728549 458 20 283 YKL103C 747 760 763 549 791 20 285 YNL090W 757 760 783549 403 20 287 YGR161C 742 760 753 549 225 20 289 YDR071C 737 757 793610 707 16

Metabolic Analysis of Transgenic Plants

The described metabolic changes in transgenic plants were identifiedusing the following experimental procedure:

a) Growth and Treatment of Plants

Plants were grown in climate chambers under standard conditions on potsoil for three weeks (see above). Eight days prior to harvest, water waswithheld for part of the plants (8-day treatment). Four days prior toharvest, water was withheld for another group of plants (4-daytreatment). The plants of “control treatment” were normally wateredthroughout the growth period. Plants due to be analysed in the sameanalytical sequence were grown side-by-side to avoid environmentalinfluences.

b) Sampling and Storage of Samples

Sampling took place in the climate chamber. Green parts were cut with apair of scissors, quickly weighed, and immediately put into a liquidnitrogen pre-cooled extraction thimble. Racks with extraction thimbleswere stored at −80° C. until extraction.

c) Freeze-Drying

Plants were not allowed to thaw or reach temperatures >−40° C. untileither the first contact with solvents or the removal of water byfreeze-drying.

The sample rack with extraction thimbles was put into the pre-cooled(−40° C.) freeze-dryer. The starting temperature for the main dryingphase was −35° C., pressure was 0.120 mbar. For the drying process,parameters were changed according to a pressure and temperature program.The final temperature (after 12 hours) was +30° C., pressure was0.001-0.004 mbar. After shutting down the vacuum pump and coolingmachine, the system was aired with dried air or Argon.

d) Extraction

Extraction thimbles with plant material were transferred to 5 mLextraction cells on the ASE (Accelerated Solvent Extractor ASE 200 withSolvent Controller and AutoASE-Software (DIONEX)) immediately afterfreeze-drying.

Polar substances were extracted with approximately 10 mL Methanol/Water(80/20, v/v) at T=70° C. und p=140 bar, 5 min heating phase, 1 minstatic Extraction. Lipid substances were extracted with approximately 10mL Methanol/Dichlormethan (40/60, v/v) at T=70° C. und p=140 bar, 5 minheating phase, 1 min static Extraction. Both extracts were collected inone extraction vial (Centrifuge tubes, 50 mL with screw-on lid andSeptum for ASE (DIONEX)).

The following internal standards were added to the extracts:LC-Standards L-Methionine-d₃, Boc-Ala-Gly-Gly-Gly-OH, L-Tryptophan-d5,Arginine¹³C₆ ¹⁵N₄, CoEnzyme Q1,2,4 and ribitol, L-glycine-2,2-d2.L-alanine-2,3,3,3-d4, alpha-methyl-glucopyranoside, nonadecanoic acidmethyl ester, undecanoic acid methyl ester, tridecanoic acid,pentadecanoic acid, nonacosanoic acid. To the resulting mixture, 8 mLwater were added. The solid residues of plant and extraction thimblewere discarded.

The extract was centrifuged at 1400 g for 5-10 minutes to speed-upphase-separation. For GC and LC analysis, 1 mL each was taken from thecolourless methanol/water upper (polar) phase. The remaining upper phasewas discarded. Of the dark-green, organic bottom phase 0.5 mL was takenfor GC and LC analysis, respectively. All sample aliquots wereevaporated using a IR-Dancer Infrared vacuum evaporator (Hettich), usinga temperature maximum of 40° C. and a maximum pressure of 10 mbar.

e) LC/MS- and LC/MS/MS-Analysis

HPLC mobile phase was added to the lipid and polar residues,respectively (volume adjusted to the weighted sample) and an HPLCanalysis using gradient elution was performed.

f) Derivatisation of the lipid phase for GC/MS-Analysis

For transmethanolysis, a mixture of 140 μl chloroform, 38 μL HCl (37%HCl in water), 320 μl methanol and 20 μl toluol was added to theresidue. The sample container was carefully closed and reaction wascarried out at 100° C. for 2 hours. Subsequently, the solution wasevaporated and the pellet was dried completely.

The methoximation of carbonyl groups was achieved by a reaction with 100μL methoxyamine-hydrochloride (5 mg/mL in Pyridine) for 1.5 hours at 60°C., in a closed vial. 20 μL of a mixture of linear, odd-numbered fattyacids was added to provide a time standard. Finally, derivatisation with100 μL N-Methyl-N-(trimethylsilyl)-2,2,2-trifluoracetamide (MSTFA) tookplace in a closed vial for 30 minutes at 60° C. The final volume for GCinjection was 220 μl.

G) Derivatisation of the Polar Phase for GC/MS-Analysis

The methoximation of carbonyl groups was achieved by a reaction with 50μL methoxyamine-hydrochloride (5 mg/mL in Pyridine) for 1.5 hours at 60°C., in a closed vial. 10 μL of a mixture of linear, odd-numbered fattyacids was added to provide a time standard. Finally, derivatisation with50 μL N-methyl-N-(trimethylsilyl)-2,2,2-trifluoracetamide (MSTFA) tookplace in a closed vial for 30 minutes at 60° C. The final volume for GCinjection was 110 μl.

h) Analysis of Different Plant Samples

Samples were measured in sequences of 20. Each sequence contained 5 wildtype and 5 transgenic plants grown under control conditions, as well as5 wild type and 5 transgenic plants from either the 4 day or 8 daydrought treatment.

The peak height or peak area of each analyte (metabolite) was dividedthrough the peak area of the respective internal standards. Data wasnormalised using the individual sample fresh weight. The resultingvalues were divided by the mean values found for wild type plants grownunder control conditions and analysed in the same sequence, resulting inthe so-called X-folds or ratios (see table 7-14), which represent valuesindependent of the analytical sequence. These ratios indicate thebehavior of the metabolite concentration of the target plants incomparison to the concentration in the wild type control plants.

In table 6 the results of the metabolite screening for the plantstransformed in genes YCL027W, YBR112c, YNL079C, YER137c, YKL103C,YNLO90W, YGR161c, YDR071c are shown.

TABLES 6 Details on screening of metabolic activity. wild typemetabolite control 4 days 8 days control 4 days 8 days YDR071C2,3-dimethyl-5-phytylquinol 1.00 0.50 0.54 2-hydroxy-palmitic acid 1.001.11 1.25 1.18 3,4-dihydroxyphenylalanine (=dopa) 1.00 1.83 3.373-hydroxy-palmitic acid 1.00 5-oxoproline 1.00 1.56 1.81 alanine 1.000.64 0.64 alpha linolenic acid (c18:3 (c9, c12, c15)) 1.00 1.24 1.40alpha-tocopherol 1.00 1.05 1.14 aminoadipic acid 1.00 1.73 1.65anhydroglucose 1.00 1.02 1.16 arginine 1.00 0.39 0.33 aspartic acid 1.002.72 2.96 beta-apo-8′ carotenal 1.00 1.21 1.22 beta-carotene 1.00 1.251.26 beta-sitosterol 1.00 1.39 1.51 beta-tocopherol 1.00 0.54 0.60palmitic acid 1.00 1.07 1.25 delta-7-cis,10-cis-hexadecadienic acid 1.001.01 1.04 (c16:2 me) hexadecatrienic acid (c16:3 me) 1.00 1.19 1.29margaric acid (c17:0 me) 1.00 1.19 1.28 delta-15-cis-tetracosenic acid(c24:1 me) 1.00 1.16 1.34 campesterol 1.00 1.32 1.65 cerotic acid(c26:0) 1.00 0.74 1.00 citrulline 1.00 0.51 0.33 cryptoxanthine 1.001.00 0.90 eicosenoic acid (20:1) 1.00 0.81 0.81 ferulic acid 1.00 1.371.47 fructose 1.00 14.10 19.78 fumarate 1.00 5.19 9.33 galactose 1.001.29 1.42 0.98 g-aminobutyric acid 1.00 1.16 1.32 gamma-tocopherol 1.000.54 0.60 gluconic acid 1.00 2.24 3.08 glucose 1.00 14.97 20.73glutamine 1.00 0.98 1.17 glutamic acid 1.00 2.01 2.69 glycerate 1.001.87 2.04 glycerinaldehyd 1.00 1.11 1.12 glycerol 1.00 1.22 1.29glycerol-3-phosphate 1.00 1.86 2.41 glycine 1.00 0.25 0.26 homoserine1.00 0.61 0.69 inositol 1.00 4.19 6.32 isoleucine 1.00 1.28 1.66iso-maltose 1.00 2.63 3.02 isopentenyl pyrophosphate 1.00 1.80 2.62leucine 1.00 1.34 1.74 lignoceric acid (c24:0) 1.00 0.91 1.02 linoleicacid (c18:2 (c9, c12)) 1.00 1.19 1.38 luteine 1.00 1.26 1.33 malate 1.002.91 3.46 mannose 1.00 16.40 17.80 triacontanoic acid 1.00 1.26 1.23methionine 1.00 1.13 1.12 methylgalactofuranoside 1.00 1.24 1.49methylgalactopyranoside 1.00 1.25 1.36 ornithine palmitic acid (c16:0)1.00 1.16 1.38 phenylalanine 1.00 0.92 1.10 phosphate 1.00 2.15 2.73proline 1.00 1.23 4.77 putrescine 1.00 0.39 pyruvate 1.00 1.30 1.21raffinose 1.00 17.04 74.78 ribonic acid 1.00 2.32 3.43 serine 1.00 0.981.13 shikimate 1.00 1.11 1.07 sinapine acid 1.00 2.74 3.44 stearic acid(c18:0) 1.00 1.18 1.35 succinate 1.00 2.98 3.49 sucrose 1.00 1.42 1.70threonine 1.00 1.26 1.68 tryptophane 1.00 1.13 1.89 tyrosine 1.00 1.562.01 ubichinone 1.00 1.02 1.20 udp-glucose 1.00 1.53 1.94 valine 1.000.98 1.18 zeaxanthine 1.00 1.27 1.34 YER137C 2,3-dimethyl-5-phytylquinol1.00 0.50 0.54 2.15 5.57 2-hydroxy-palmitic acid 1.00 1.11 1.253,4-dihydroxyphenylalanine (=dopa) 1.00 1.83 3.37 3-hydroxy-palmiticacid 1.00 5-oxoproline 1.00 1.56 1.81 1.55 1.73 alanine 1.00 0.64 0.641.32 0.94 alpha linolenic acid (c18:3 (c9, c12, c15)) 1.00 1.24 1.40alpha-tocopherol 1.00 1.05 1.14 aminoadipic acid 1.00 1.73 1.65anhydroglucose 1.00 1.02 1.16 1.26 0.63 arginine 1.00 0.39 0.33 asparticacid 1.00 2.72 2.96 beta-apo-8′ carotenal 1.00 1.21 1.22 beta-carotene1.00 1.25 1.26 beta-sitosterol 1.00 1.39 1.51 0.88 1.17 1.35beta-tocopherol 1.00 0.54 0.60 2.78 5.83 palmitic acid 1.00 1.07 1.25delta-7-cis,10-cis-hexadecadienic acid 1.00 1.01 1.04 (c16:2 me)hexadecatrienic acid (c16:3 me) 1.00 1.19 1.29 margaric acid (c17:0 me)1.00 1.19 1.28 delta-15-cis-tetracosenic acid (c24:1 me) 1.00 1.16 1.34campesterol 1.00 1.32 1.65 1.10 1.28 cerotic acid (c26:0) 1.00 0.74 1.003.24 1.78 citrulline 1.00 0.51 0.33 cryptoxanthine 1.00 1.00 0.90eicosenoic acid (20:1) 1.00 0.81 0.81 ferulic acid 1.00 1.37 1.47fructose 1.00 14.10 19.78 fumarate 1.00 5.19 9.33 galactose 1.00 1.291.42 g-aminobutyric acid 1.00 1.16 1.32 4.44 10.51 gamma-tocopherol 1.000.54 0.60 2.78 5.83 gluconic acid 1.00 2.24 3.08 glucose 1.00 14.9720.73 glutamine 1.00 0.98 1.17 glutamic acid 1.00 2.01 2.69 glycerate1.00 1.87 2.04 1.17 2.32 glycerinaldehyd 1.00 1.11 1.12 glycerol 1.001.22 1.29 glycerol-3-phosphate 1.00 1.86 2.41 1.12 1.57 2.05 glycine1.00 0.25 0.26 homoserine 1.00 0.61 0.69 inositol 1.00 4.19 6.32isoleucine 1.00 1.28 1.66 iso-maltose 1.00 2.63 3.02 isopentenylpyrophosphate 1.00 1.80 2.62 leucine 1.00 1.34 1.74 lignoceric acid(c24:0) 1.00 0.91 1.02 2.01 1.74 2.03 linoleic acid (c18:2 (c9, c12))1.00 1.19 1.38 luteine 1.00 1.26 1.33 malate 1.00 2.91 3.46 1.67 5.706.01 mannose 1.00 16.40 17.80 triacontanoic acid 1.00 1.26 1.23methionine 1.00 1.13 1.12 methylgalactofuranoside 1.00 1.24 1.49methylgalactopyranoside 1.00 1.25 1.36 ornithine palmitic acid (c16:0)1.00 1.16 1.38 phenylalanine 1.00 0.92 1.10 phosphate 1.00 2.15 2.73proline 1.00 1.23 4.77 putrescine 1.00 0.39 pyruvate 1.00 1.30 1.21raffinose 1.00 17.04 74.78 ribonic acid 1.00 2.32 3.43 serine 1.00 0.981.13 shikimate 1.00 1.11 1.07 1.40 1.50 sinapine acid 1.00 2.74 3.44stearic acid (c18:0) 1.00 1.18 1.35 succinate 1.00 2.98 3.49 sucrose1.00 1.42 1.70 threonine 1.00 1.26 1.68 tryptophane 1.00 1.13 1.89tyrosine 1.00 1.56 2.01 ubichinone 1.00 1.02 1.20 udp-glucose 1.00 1.531.94 valine 1.00 0.98 1.18 zeaxanthine 1.00 1.27 1.34 YBR112C2,3-dimethyl-5-phytylquinol 1.00 0.50 0.54 0.63 2-hydroxy-palmitic acid1.00 1.11 1.25 3,4-dihydroxyphenylalanine (=dopa) 1.00 1.83 3.373-hydroxy-palmitic acid 1.00 5-oxoproline 1.00 1.56 1.81 alanine 1.000.64 0.64 alpha linolenic acid (c18:3 (c9, c12, c15)) 1.00 1.24 1.401.12 1.42 alpha-tocopherol 1.00 1.05 1.14 aminoadipic acid 1.00 1.731.65 3.90 12.95 anhydroglucose 1.00 1.02 1.16 arginine 1.00 0.39 0.33aspartic acid 1.00 2.72 2.96 beta-apo-8′ carotenal 1.00 1.21 1.22beta-carotene 1.00 1.25 1.26 1.35 beta-sitosterol 1.00 1.39 1.51 1.19beta-tocopherol 1.00 0.54 0.60 0.70 palmitic acid 1.00 1.07 1.25delta-7-cis,10-cis-hexadecadienic acid 1.00 1.01 1.04 (c16:2 me)hexadecatrienic acid (c16:3 me) 1.00 1.19 1.29 margaric acid (c17:0 me)1.00 1.19 1.28 delta-15-cis-tetracosenic acid (c24:1 me) 1.00 1.16 1.34campesterol 1.00 1.32 1.65 1.42 cerotic acid (c26:0) 1.00 0.74 1.00citrulline 1.00 0.51 0.33 cryptoxanthine 1.00 1.00 0.90 eicosenoic acid(20:1) 1.00 0.81 0.81 ferulic acid 1.00 1.37 1.47 1.20 fructose 1.0014.10 19.78 fumarate 1.00 5.19 9.33 galactose 1.00 1.29 1.42g-aminobutyric acid 1.00 1.16 1.32 gamma-tocopherol 1.00 0.54 0.60 0.70gluconic acid 1.00 2.24 3.08 glucose 1.00 14.97 20.73 glutamine 1.000.98 1.17 glutamic acid 1.00 2.01 2.69 glycerate 1.00 1.87 2.04glycerinaldehyd 1.00 1.11 1.12 glycerol 1.00 1.22 1.29glycerol-3-phosphate 1.00 1.86 2.41 glycine 1.00 0.25 0.26 homoserine1.00 0.61 0.69 inositol 1.00 4.19 6.32 isoleucine 1.00 1.28 1.66iso-maltose 1.00 2.63 3.02 isopentenyl pyrophosphate 1.00 1.80 2.62leucine 1.00 1.34 1.74 lignoceric acid (c24:0) 1.00 0.91 1.02 linoleicacid (c18:2 (c9, c12)) 1.00 1.19 1.38 luteine 1.00 1.26 1.33 1.17 malate1.00 2.91 3.46 mannose 1.00 16.40 17.80 triacontanoic acid 1.00 1.261.23 methionine 1.00 1.13 1.12 methylgalactofuranoside 1.00 1.24 1.49methylgalactopyranoside 1.00 1.25 1.36 ornithine palmitic acid (c16:0)1.00 1.16 1.38 1.11 phenylalanine 1.00 0.92 1.10 phosphate 1.00 2.152.73 proline 1.00 1.23 4.77 putrescine 1.00 0.39 pyruvate 1.00 1.30 1.21raffinose 1.00 17.04 74.78 ribonic acid 1.00 2.32 3.43 serine 1.00 0.981.13 shikimate 1.00 1.11 1.07 sinapine acid 1.00 2.74 3.44 stearic acid(c18:0) 1.00 1.18 1.35 succinate 1.00 2.98 3.49 sucrose 1.00 1.42 1.70threonine 1.00 1.26 1.68 tryptophane 1.00 1.13 1.89 tyrosine 1.00 1.562.01 ubichinone 1.00 1.02 1.20 udp-glucose 1.00 1.53 1.94 valine 1.000.98 1.18 zeaxanthine 1.00 1.27 1.34 YGR161C 2,3-dimethyl-5-phytylquinol1.00 0.50 0.54 2-hydroxy-palmitic acid 1.00 1.11 1.253,4-dihydroxyphenylalanine (=dopa) 1.00 1.83 3.37 3-hydroxy-palmiticacid 1.00 5-oxoproline 1.00 1.56 1.81 alanine 1.00 0.64 0.64 alphalinolenic acid (c18:3 (c9, c12, c15)) 1.00 1.24 1.40 alpha-tocopherol1.00 1.05 1.14 aminoadipic acid 1.00 1.73 1.65 anhydroglucose 1.00 1.021.16 arginine 1.00 0.39 0.33 aspartic acid 1.00 2.72 2.96 beta-apo-8′carotenal 1.00 1.21 1.22 beta-carotene 1.00 1.25 1.26 beta-sitosterol1.00 1.39 1.51 beta-tocopherol 1.00 0.54 0.60 palmitic acid 1.00 1.071.25 delta-7-cis,10-cis-hexadecadienic acid 1.00 1.01 1.04 1.14 (c16:2me) hexadecatrienic acid (c16:3 me) 1.00 1.19 1.29 margaric acid (c17:0me) 1.00 1.19 1.28 1.22 delta-15-cis-tetracosenic acid (c24:1 me) 1.001.16 1.34 campesterol 1.00 1.32 1.65 cerotic acid (c26:0) 1.00 0.74 1.00citrulline 1.00 0.51 0.33 cryptoxanthine 1.00 1.00 0.90 eicosenoic acid(20:1) 1.00 0.81 0.81 ferulic acid 1.00 1.37 1.47 fructose 1.00 14.1019.78 fumarate 1.00 5.19 9.33 galactose 1.00 1.29 1.42 1.15g-aminobutyric acid 1.00 1.16 1.32 gamma-tocopherol 1.00 0.54 0.60gluconic acid 1.00 2.24 3.08 glucose 1.00 14.97 20.73 glutamine 1.000.98 1.17 glutamic acid 1.00 2.01 2.69 glycerate 1.00 1.87 2.04glycerinaldehyd 1.00 1.11 1.12 glycerol 1.00 1.22 1.29glycerol-3-phosphate 1.00 1.86 2.41 glycine 1.00 0.25 0.26 homoserine1.00 0.61 0.69 inositol 1.00 4.19 6.32 isoleucine 1.00 1.28 1.66iso-maltose 1.00 2.63 3.02 isopentenyl pyrophosphate 1.00 1.80 2.62leucine 1.00 1.34 1.74 lignoceric acid (c24:0) 1.00 0.91 1.02 linoleicacid (c18:2 (c9, c12)) 1.00 1.19 1.38 luteine 1.00 1.26 1.33 malate 1.002.91 3.46 mannose 1.00 16.40 17.80 triacontanoic acid 1.00 1.26 1.23methionine 1.00 1.13 1.12 methylgalactofuranoside 1.00 1.24 1.49methylgalactopyranoside 1.00 1.25 1.36 1.18 ornithine palmitic acid(c16:0) 1.00 1.16 1.38 phenylalanine 1.00 0.92 1.10 phosphate 1.00 2.152.73 proline 1.00 1.23 4.77 putrescine 1.00 0.39 pyruvate 1.00 1.30 1.21raffinose 1.00 17.04 74.78 ribonic acid 1.00 2.32 3.43 serine 1.00 0.981.13 shikimate 1.00 1.11 1.07 sinapine acid 1.00 2.74 3.44 stearic acid(c18:0) 1.00 1.18 1.35 succinate 1.00 2.98 3.49 sucrose 1.00 1.42 1.70threonine 1.00 1.26 1.68 tryptophane 1.00 1.13 1.89 tyrosine 1.00 1.562.01 ubichinone 1.00 1.02 1.20 udp-glucose 1.00 1.53 1.94 valine 1.000.98 1.18 zeaxanthine 1.00 1.27 1.34 YKL103C 2,3-dimethyl-5-phytylquinol1.00 0.50 0.54 2-hydroxy-palmitic acid 1.00 1.11 1.253,4-dihydroxyphenylalanine (=dopa) 1.00 1.83 3.37 3-hydroxy-palmiticacid 1.00 5-oxoproline 1.00 1.56 1.81 alanine 1.00 0.64 0.64 alphalinolenic acid (c18:3 (c9, c12, c15)) 1.00 1.24 1.40 alpha-tocopherol1.00 1.05 1.14 aminoadipic acid 1.00 1.73 1.65 anhydroglucose 1.00 1.021.16 arginine 1.00 0.39 0.33 aspartic acid 1.00 2.72 2.96 beta-apo-8′carotenal 1.00 1.21 1.22 beta-carotene 1.00 1.25 1.26 beta-sitosterol1.00 1.39 1.51 beta-tocopherol 1.00 0.54 0.60 palmitic acid 1.00 1.071.25 delta-7-cis,10-cis-hexadecadienic acid 1.00 1.01 1.04 1.18 1.161.30 (c16:2 me) hexadecatrienic acid (c16:3 me) 1.00 1.19 1.29 margaricacid (c17:0 me) 1.00 1.19 1.28 delta-15-cis-tetracosenic acid (c24:1 me)1.00 1.16 1.34 campesterol 1.00 1.32 1.65 cerotic acid (c26:0) 1.00 0.741.00 citrulline 1.00 0.51 0.33 cryptoxanthine 1.00 1.00 0.90 eicosenoicacid (20:1) 1.00 0.81 0.81 ferulic acid 1.00 1.37 1.47 fructose 1.0014.10 19.78 fumarate 1.00 5.19 9.33 galactose 1.00 1.29 1.42g-aminobutyric acid 1.00 1.16 1.32 gamma-tocopherol 1.00 0.54 0.60gluconic acid 1.00 2.24 3.08 glucose 1.00 14.97 20.73 glutamine 1.000.98 1.17 glutamic acid 1.00 2.01 2.69 glycerate 1.00 1.87 2.04glycerinaldehyd 1.00 1.11 1.12 glycerol 1.00 1.22 1.29glycerol-3-phosphate 1.00 1.86 2.41 glycine 1.00 0.25 0.26 homoserine1.00 0.61 0.69 inositol 1.00 4.19 6.32 isoleucine 1.00 1.28 1.66iso-maltose 1.00 2.63 3.02 isopentenyl pyrophosphate 1.00 1.80 2.62leucine 1.00 1.34 1.74 lignoceric acid (c24:0) 1.00 0.91 1.02 linoleicacid (c18:2 (c9, c12)) 1.00 1.19 1.38 luteine 1.00 1.26 1.33 malate 1.002.91 3.46 mannose 1.00 16.40 17.80 triacontanoic acid 1.00 1.26 1.23methionine 1.00 1.13 1.12 methylgalactofuranoside 1.00 1.24 1.49methylgalactopyranoside 1.00 1.25 1.36 ornithine 1.70 1.52 palmitic acid(c16:0) 1.00 1.16 1.38 phenylalanine 1.00 0.92 1.10 phosphate 1.00 2.152.73 proline 1.00 1.23 4.77 putrescine 1.00 0.39 pyruvate 1.00 1.30 1.21raffinose 1.00 17.04 74.78 ribonic acid 1.00 2.32 3.43 serine 1.00 0.981.13 shikimate 1.00 1.11 1.07 sinapine acid 1.00 2.74 3.44 stearic acid(c18:0) 1.00 1.18 1.35 succinate 1.00 2.98 3.49 sucrose 1.00 1.42 1.70threonine 1.00 1.26 1.68 tryptophane 1.00 1.13 1.89 tyrosine 1.00 1.562.01 ubichinone 1.00 1.02 1.20 udp-glucose 1.00 1.53 1.94 valine 1.000.98 1.18 zeaxanthine 1.00 1.27 1.34 YCL027W 2,3-dimethyl-5-phytylquinol1.00 0.50 0.54 2-hydroxy-palmitic acid 1.00 1.11 1.253,4-dihydroxyphenylalanine (=dopa) 1.00 1.83 3.37 3-hydroxy-palmiticacid 1.00 5-oxoproline 1.00 1.56 1.81 alanine 1.00 0.64 0.64 alphalinolenic acid (c18:3 (c9, c12, c15)) 1.00 1.24 1.40 alpha-tocopherol1.00 1.05 1.14 aminoadipic acid 1.00 1.73 1.65 anhydroglucose 1.00 1.021.16 arginine 1.00 0.39 0.33 aspartic acid 1.00 2.72 2.96 beta-apo-8′carotenal 1.00 1.21 1.22 beta-carotene 1.00 1.25 1.26 beta-sitosterol1.00 1.39 1.51 beta-tocopherol 1.00 0.54 0.60 palmitic acid 1.00 1.071.25 delta-7-cis,10-cis-hexadecadienic acid 1.00 1.01 1.04 (c16:2 me)hexadecatrienic acid (c16:3 me) 1.00 1.19 1.29 margaric acid (c17:0 me)1.00 1.19 1.28 delta-15-cis-tetracosenic acid (c24:1 me) 1.00 1.16 1.34campesterol 1.00 1.32 1.65 cerotic acid (c26:0) 1.00 0.74 1.00citrulline 1.00 0.51 0.33 cryptoxanthine 1.00 1.00 0.90 eicosenoic acid(20:1) 1.00 0.81 0.81 ferulic acid 1.00 1.37 1.47 0.79 0.74 0.80fructose 1.00 14.10 19.78 fumarate 1.00 5.19 9.33 galactose 1.00 1.291.42 g-aminobutyric acid 1.00 1.16 1.32 gamma-tocopherol 1.00 0.54 0.60gluconic acid 1.00 2.24 3.08 glucose 1.00 14.97 20.73 glutamine 1.000.98 1.17 glutamic acid 1.00 2.01 2.69 glycerate 1.00 1.87 2.04glycerinaldehyd 1.00 1.11 1.12 glycerol 1.00 1.22 1.29glycerol-3-phosphate 1.00 1.86 2.41 glycine 1.00 0.25 0.26 homoserine1.00 0.61 0.69 inositol 1.00 4.19 6.32 isoleucine 1.00 1.28 1.66iso-maltose 1.00 2.63 3.02 isopentenyl pyrophosphate 1.00 1.80 2.62leucine 1.00 1.34 1.74 lignoceric acid (c24:0) 1.00 0.91 1.02 linoleicacid (c18:2 (c9, c12)) 1.00 1.19 1.38 luteine 1.00 1.26 1.33 malate 1.002.91 3.46 mannose 1.00 16.40 17.80 triacontanoic acid 1.00 1.26 1.23methionine 1.00 1.13 1.12 methylgalactofuranoside 1.00 1.24 1.49methylgalactopyranoside 1.00 1.25 1.36 ornithine palmitic acid (c16:0)1.00 1.16 1.38 phenylalanine 1.00 0.92 1.10 phosphate 1.00 2.15 2.73proline 1.00 1.23 4.77 putrescine 1.00 0.39 pyruvate 1.00 1.30 1.21raffinose 1.00 17.04 74.78 ribonic acid 1.00 2.32 3.43 serine 1.00 0.981.13 shikimate 1.00 1.11 1.07 sinapine acid 1.00 2.74 3.44 stearic acid(c18:0) 1.00 1.18 1.35 succinate 1.00 2.98 3.49 sucrose 1.00 1.42 1.70threonine 1.00 1.26 1.68 tryptophane 1.00 1.13 1.89 tyrosine 1.00 1.562.01 ubichinone 1.00 1.02 1.20 udp-glucose 1.00 1.53 1.94 valine 1.000.98 1.18 zeaxanthine 1.00 1.27 1.34 YNL079C 2,3-dimethyl-5-phytylquinol1.00 0.50 0.54 0.59 2-hydroxy-palmitic acid 1.00 1.11 1.253,4-dihydroxyphenylalanine (=dopa) 1.00 1.83 3.37 3-hydroxy-palmiticacid 1.00 ! 5-oxoproline 1.00 1.56 1.81 alanine 1.00 0.64 0.64 alphalinolenic acid (c18:3 (c9, c12, c15)) 1.00 1.24 1.40 1.53 1.56alpha-tocopherol 1.00 1.05 1.14 2.64 3.66 aminoadipic acid 1.00 1.731.65 anhydroglucose 1.00 1.02 1.16 arginine 1.00 0.39 0.33 aspartic acid1.00 2.72 2.96 beta-apo-8′ carotenal 1.00 1.21 1.22 beta-carotene 1.001.25 1.26 1.26 1.20 beta-sitosterol 1.00 1.39 1.51 beta-tocopherol 1.000.54 0.60 0.73 0.56 palmitic acid 1.00 1.07 1.25delta-7-cis,10-cis-hexadecadienic acid 1.00 1.01 1.04 (c16:2 me)hexadecatrienic acid (c16:3 me) 1.00 1.19 1.29 margaric acid (c17:0 me)1.00 1.19 1.28 1.22 1.53 1.36 delta-15-cis-tetracosenic acid (c24:1 me)1.00 1.16 1.34 campesterol 1.00 1.32 1.65 1.79 1.82 cerotic acid (c26:0)1.00 0.74 1.00 citrulline 1.00 0.51 0.33 cryptoxanthine 1.00 1.00 0.90eicosenoic acid (20:1) 1.00 0.81 0.81 ferulic acid 1.00 1.37 1.47fructose 1.00 14.10 19.78 fumarate 1.00 5.19 9.33 galactose 1.00 1.291.42 g-aminobutyric acid 1.00 1.16 1.32 gamma-tocopherol 1.00 0.54 0.60gluconic acid 1.00 2.24 3.08 glucose 1.00 14.97 20.73 glutamine 1.000.98 1.17 glutamic acid 1.00 2.01 2.69 glycerate 1.00 1.87 2.04glycerinaldehyd 1.00 1.11 1.12 glycerol 1.00 1.22 1.29glycerol-3-phosphate 1.00 1.86 2.41 glycine 1.00 0.25 0.26 homoserine1.00 0.61 0.69 inositol 1.00 4.19 6.32 4.73 6.12 isoleucine 1.00 1.281.66 iso-maltose 1.00 2.63 3.02 4.42 4.93 isopentenyl pyrophosphate 1.001.80 2.62 leucine 1.00 1.34 1.74 1.51 2.19 lignoceric acid (c24:0) 1.000.91 1.02 linoleic acid (c18:2 (c9, c12)) 1.00 1.19 1.38 luteine 1.001.26 1.33 malate 1.00 2.91 3.46 1.49 5.72 mannose 1.00 16.40 17.80triacontanoic acid 1.00 1.26 1.23 methionine 1.00 1.13 1.12methylgalactofuranoside 1.00 1.24 1.49 methylgalactopyranoside 1.00 1.251.36 ornithine palmitic acid (c16:0) 1.00 1.16 1.38 phenylalanine 1.000.92 1.10 phosphate 1.00 2.15 2.73 proline 1.00 1.23 4.77 putrescine1.00 0.39 pyruvate 1.00 1.30 1.21 raffinose 1.00 17.04 74.78 ribonicacid 1.00 2.32 3.43 serine 1.00 0.98 1.13 shikimate 1.00 1.11 1.07sinapine acid 1.00 2.74 3.44 3.89 2.79 stearic acid (c18:0) 1.00 1.181.35 succinate 1.00 2.98 3.49 sucrose 1.00 1.42 1.70 threonine 1.00 1.261.68 tryptophane 1.00 1.13 1.89 2.68 2.98 tyrosine 1.00 1.56 2.01ubichinone 1.00 1.02 1.20 0.88 1.02 udp-glucose 1.00 1.53 1.94 valine1.00 0.98 1.18 zeaxanthine 1.00 1.27 1.34 YNL090W2,3-dimethyl-5-phytylquinol 1.00 0.50 0.54 2-hydroxy-palmitic acid 1.001.11 1.25 1.15 1.25 3,4-dihydroxyphenylalanine (=dopa) 1.00 1.83 3.373-hydroxy-palmitic acid 1.00 5-oxoproline 1.00 1.56 1.81 alanine 1.000.64 0.64 alpha linolenic acid (c18:3 (c9, c12, c15)) 1.00 1.24 1.40alpha-tocopherol 1.00 1.05 1.14 1.30 5.28 aminoadipic acid 1.00 1.731.65 anhydroglucose 1.00 1.02 1.16 arginine 1.00 0.39 0.33 0.85 asparticacid 1.00 2.72 2.96 beta-apo-8′ carotenal 1.00 1.21 1.22 beta-carotene1.00 1.25 1.26 1.15 1.91 beta-sitosterol 1.00 1.39 1.51 beta-tocopherol1.00 0.54 0.60 palmitic acid 1.00 1.07 1.25delta-7-cis,10-cis-hexadecadienic acid 1.00 1.01 1.04 (c16:2 me)hexadecatrienic acid (c16:3 me) 1.00 1.19 1.29 margaric acid (c17:0 me)1.00 1.19 1.28 delta-15-cis-tetracosenic acid (c24:1 me) 1.00 1.16 1.34campesterol 1.00 1.32 1.65 cerotic acid (c26:0) 1.00 0.74 1.00 2.98 3.93citrulline 1.00 0.51 0.33 cryptoxanthine 1.00 1.00 0.90 eicosenoic acid(20:1) 1.00 0.81 0.81 ferulic acid 1.00 1.37 1.47 fructose 1.00 14.1019.78 fumarate 1.00 5.19 9.33 0.76 galactose 1.00 1.29 1.42g-aminobutyric acid 1.00 1.16 1.32 gamma-tocopherol 1.00 0.54 0.60 1.69gluconic acid 1.00 2.24 3.08 glucose 1.00 14.97 20.73 glutamine 1.000.98 1.17 glutamic acid 1.00 2.01 2.69 glycerate 1.00 1.87 2.04glycerinaldehyd 1.00 1.11 1.12 glycerol 1.00 1.22 1.29glycerol-3-phosphate 1.00 1.86 2.41 glycine 1.00 0.25 0.26 homoserine1.00 0.61 0.69 inositol 1.00 4.19 6.32 1.46 9.70 isoleucine 1.00 1.281.66 3.54 4.86 iso-maltose 1.00 2.63 3.02 3.59 20.72 14.65 isopentenylpyrophosphate 1.00 1.80 2.62 1.79 leucine 1.00 1.34 1.74 lignoceric acid(c24:0) 1.00 0.91 1.02 linoleic acid (c18:2 (c9, c12)) 1.00 1.19 1.38luteine 1.00 1.26 1.33 malate 1.00 2.91 3.46 2.12 14.49 mannose 1.0016.40 17.80 triacontanoic acid 1.00 1.26 1.23 methionine 1.00 1.13 1.12methylgalactofuranoside 1.00 1.24 1.49 methylgalactopyranoside 1.00 1.251.36 ornithine palmitic acid (c16:0) 1.00 1.16 1.38 phenylalanine 1.000.92 1.10 phosphate 1.00 2.15 2.73 proline 1.00 1.23 4.77 putrescine1.00 0.39 pyruvate 1.00 1.30 1.21 raffinose 1.00 17.04 74.78 ribonicacid 1.00 2.32 3.43 serine 1.00 0.98 1.13 shikimate 1.00 1.11 1.07sinapine acid 1.00 2.74 3.44 stearic acid (c18:0) 1.00 1.18 1.35succinate 1.00 2.98 3.49 sucrose 1.00 1.42 1.70 threonine 1.00 1.26 1.68tryptophane 1.00 1.13 1.89 tyrosine 1.00 1.56 2.01 ubichinone 1.00 1.021.20 udp-glucose 1.00 1.53 1.94 valine 1.00 0.98 1.18 1.13 1.55 1.80zeaxanthine 1.00 1.27 1.34

Example 2 Engineering Stress-Tolerant Arabidopsis Plants byOver-Expressing Stress Related Protein Encoding Genes from SaccharomycesCereviesae or E. Coli Or Brassica Napus, Glycine Max, and Oryza SativaUsing Stress-Inducible and Tissue-Specific Promoters

Transgenic Arabidopsis plants were created as in example 1 to expressthe stress related protein encoding transgenes under the control ofeither a tissue-specific or stress-inducible promoter. Constitutiveexpression of a transgene may cause deleterious side effects. Stressinducible expression was achieved using promoters selected from thoselisted above in Table 1.

T2 generation plants were produced and treated with drought stress intwo experiments. For the first drought experiment, the plants weredeprived of water until the plant and soil were desiccated. At varioustimes after withholding water, a normal watering schedule was resumedand the plants were grown to maturity. Seed yield was determined asseeds per plant. At an equivalent degree of drought stress, tolerantplants were able to resume normal growth and produced more seeds thannon-transgenic control plants. Proline content of the leaves andstomatal aperture were also measured at various times during the droughtstress. Tolerant plants maintained a lower proline content and a greaterstomatal aperture than the non-transgenic control plants.

An alternative method to impose water stress on the transgenic plantswas by treatment with water containing an osmolyte such as polyethyleneglycol (PEG) at specific water potential. Since PEG may be toxic, theplants were given only a short term exposure and then normal wateringwas resumed. As above, seed yields were measured from the mature plants.The response was measured during the stress period by physicalmeasurements, such as stomatal aperture or osmotic potential, orbiochemical measurements, such as accumulation of proline. Tolerantplants had higher seed yields, maintained their stomatal aperture andshowed only slight changes in osmotic potential and proline levels,whereas the susceptible non-transgenic control plants closed theirstomata and exhibited increased osmotic potential and proline levels.

The transgenic plants with a constitutive promoter controllingtranscription of the transgene were compared to those plants with adrought-inducible promoter in the absence of stress. The resultsindicated that the metabolite and gene expression changes did not occurwhen plants with the stress-inducible promoter were grown in the absenceof stress. These plants also had higher seed yields than those with theconstitutive promoter.

Example 3 Over-Expression of Stress Related Genes from SaccharomycesCerevisiae or E. Coli or Brassica Napus, Glycine Max, and Oryza SativaProvides Tolerance of Multiple Abiotic Stresses

Plants that exhibit tolerance of one abiotic stress often exhibittolerance of another environmental stress or an oxygen free radicalgenerating herbicide. This phenomenon of cross-tolerance is notunderstood at a mechanistic level (McKersie and Leshem, 1994).Nonetheless, it is reasonable to expect that plants exhibiting enhanceddrought tolerance due to the expression of a transgene might alsoexhibit tolerance of low temperatures, freezing, salt, air pollutantssuch as ozone, and other abiotic stresses. In support of thishypothesis, the expression of several genes are up or down-regulated bymultiple abiotic stress factors including cold, salt, osmoticum, ABA,etc (e.g. Hong et al. (1992) Developmental and organ-specific expressionof an ABA- and stress-induced protein in barley. Plant Mol Biol 18:663-674; Jagendorf and Takabe (2001) Inducers of glycinebetainesynthesis in barley. Plant Physiol 127: 1827-1835); Mizoguchi et al.(1996) A gene encoding a mitogen-activated protein kinase is inducedsimultaneously with genes for a mitogen-activated protein kinase and anS6 ribosomal protein kinase by touch, cold, and water stress inArabidopsis thaliana. Proc Natl Acad Sci USA 93: 765-769; Zhu (2001)Cell signaling under salt, water and cold stresses. Curr Opin Plant Biol4: 401-406).

To determine salt tolerance, seeds of Arabidopsis thaliana weresterilized (100% bleach, 0.1% TritonX for five minutes two times andrinsed five times with ddH2O). Seeds were plated on non-selection media(½ MS, 0.6% phytagar, 0.5 g/L MES, 1% sucrose, 2 μg/ml benamyl). Seedsare allowed to germinate for approximately ten days. At the 4-5 leafstage, transgenic plants were potted into 5.5 cm diameter pots andallowed to grow (22° C., continuous light) for approximately seven days,watering as needed. To begin the assay, two liters of 100 mM NaCl and ⅛MS was added to the tray under the pots. To the tray containing thecontrol plants, three liters of ⅛ MS was added. The concentrations ofNaCl supplementation were increased stepwise by 50 mM every 4 days up to200 mM. After the salt treatment with 200 mM, fresh and dry weights ofthe plants as well as seed yields were determined.

To determine cold tolerance, seeds of the transgenic and cold lines weregerminated and grown for approximately 10 days to the 4-5 leaf stage asabove. The plants were then transferred to cold temperatures (5° C.) andgrown through the flowering and seed set stages of development.Photosynthesis was measured using chlorophyll fluorescence as anindicator of photosynthetic fitness and integrity of the photosystems.Seed yield and plant dry weight were measured as an indictor of plantbiomass production.

Plants that had tolerance to salinity or cold had higher seed yields,photosynthesis and dry matter production than susceptible plants.

Example 4 Engineering Stress-Tolerant Alfalfa Plants by Over-ExpressingStress Related Genes from Saccharomyces Cerevisiae or E. Coli orBrassica Napus, Glycine Max, and Oryza Sativa

A regenerating clone of alfalfa (Medicago sativa) was transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown DCW and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659).

Petiole explants were cocultivated with an overnight culture ofAgrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 PlantPhysiol 119: 839-847) or LBA4404 containing a binary vector. Manydifferent binary vector systems have been described for planttransformation (e.g. An, G. in Agrobacterium Protocols. Methods inMolecular Biology vol 44, pp 47-62, Gartland K M A and M R Davey eds.Humana Press, Totowa, N.J.). Many are based on the vector pBIN19described by Bevan (Nucleic Acid Research. 1984. 12:8711-8721) thatincludes a plant gene expression cassette flanked by the left and rightborder sequences from the Ti plasmid of Agrobacterium tumefaciens. Aplant gene expression cassette consists of at least two genes—aselection marker gene and a plant promoter regulating the transcriptionof the cDNA or genomic DNA of the trait gene. Various selection markergenes can be used including the Arabidopsis gene encoding a mutatedacetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. Nos. 5,767,3666 and6,225,105). Similarly, various promoters can be used to regulate thetrait gene that provides constitutive, developmental, tissue orenvironmental regulation of gene transcription. In this example, the 34Spromoter (GenBank Accession numbers M59930 and X16673) was used toprovide constitutive expression of the trait gene.

The explants were cocultivated for 3 d in the dark on SH inductionmedium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and100 μm acetosyringinone. The explants were washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos were transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos were subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings were transplantedinto pots and grown in a greenhouse.

The T0 transgenic plants were propagated by node cuttings and rooted inTurface growth medium. The plants were defoliated and grown to a heightof about cm (approximately 2 weeks after defoliation). The plants werethen subjected to drought stress in two experiments.

For the first drought experiment, the seedlings received no water for aperiod up to 3 weeks at which time the plant and soil were desiccated.At various times after withholding water, a normal watering schedule wasresumed. At one week after resuming watering, the fresh and dry weightsof the shoots was determined. At an equivalent degree of drought stress,tolerant plants were able to resume normal growth whereas susceptibleplants had died or suffered significant injury resulting in less drymatter. Proline content of the leaves and stomatal aperture were alsomeasured at various times during the drought stress. Tolerant plantsmaintained a lower proline content and a greater stomatal aperture thanthe non-transgenic control plants.

An alternative method to impose water stress on the transgenic plantswas by treatment with a solution at specific water potential, containingan osmolyte such as polyethylene glycol (PEG). The PEG treatment wasgiven to either detached leaves (e.g. Djilianov et al., 1997 PlantScience 129: 147-156) or to the roots (Wakabayashi et al., 1997 PlantPhysiol 113: 967-973). Since PEG may be toxic, the plants were givenonly a short term exposure. The response was measured as physicalmeasurements such as stomatal aperture or osmotic potential, orbiochemical measurements such as accumulation of proline. Tolerantplants maintained their stomatal aperture and showed only slight changesin osmotic potential, whereas the susceptible non-transgenic controlplants closed their stomata and exhibited increased osmotic potential.In addition the changes in proline and other metabolites were less inthe tolerant transgenic plants than in the non-transgenic controlplants.

Tolerance of salinity and cold were measured using methods as describedin example 3. Plants that had tolerance to salinity or cold had higherseed yields, photosynthesis and dry matter production than susceptibleplants.

Example 5 Engineering Stress-Tolerant Ryegrass Plants by Over-ExpressingStress Related Genes from Saccharomyces Cerevisiae or E. Coli orBrassica Napus, Glycine Max, and Oryza Sativa

Seeds of several different ryegrass varieties may be used as explantsources for transformation, including the commercial variety Gunneavailable from Svalof Weibull seed company or the variety Affinity.Seeds were surface-sterilized sequentially with 1% Tween-20 for 1minute, 100% bleach for 60 minutes, 3 rinses with 5 minutes each withde-ionized and distilled H2O, and then germinated for 3-4 days on moist,sterile filter paper in the dark. Seedlings were further sterilized for1 minute with 1% Tween-20, 5 minutes with 75% bleach, and rinsed 3 timeswith ddH2O, min each.

Surface-sterilized seeds were placed on the callus induction mediumcontaining Murashige and Skoog basal salts and vitamins, 20 g/l sucrose,150 mg/l asparagine, 500 mg/l casein hydrolysate, 3 g/l Phytagel, 10mg/l BAP, and 5 mg/l dicamba. Plates were incubated in the dark at 25°C. for 4 weeks for seed germination and embryogenic callus induction.

After 4 weeks on the callus induction medium, the shoots and roots ofthe seedlings were trimmed away, the callus was transferred to freshmedia, maintained in culture for another 4 weeks, and then transferredto MSO medium in light for 2 weeks. Several pieces of callus (11-17weeks old) were either strained through a mesh sieve and put onto callusinduction medium, or cultured in 100 ml of liquid ryegrass callusinduction media (same medium as for callus induction with agar) in a 250ml flask. The flask was wrapped in foil and shaken at 175 rpm in thedark at 23° C. for 1 week. Sieving the liquid culture with a 40-meshsieve collected the cells. The fraction collected on the sieve wasplated and cultured on solid ryegrass callus induction medium for 1 weekin the dark at 25° C. The callus was then transferred to and cultured onMS medium containing 1% sucrose for 2 weeks.

Transformation can be accomplished with either Agrobacterium of withparticle bombardment methods. An expression vector is created containinga constitutive plant promoter and the cDNA of the gene in a pUC vector.The plasmid DNA was prepared from E. coli cells using with Qiagen kitaccording to manufacturer's instruction. Approximately 2 g ofembryogenic callus was spread in the center of a sterile filter paper ina Petri dish. An aliquot of liquid MSO with 10 g/l sucrose was added tothe filter paper. Gold particles (1.0 μm in size) were coated withplasmid DNA according to method of Sanford et al., 1993 and delivered tothe embryogenic callus with the following parameters: 500 μg particlesand 2 μg DNA per shot, 1300 psi and a target distance of 8.5 cm fromstopping plate to plate of callus and 1 shot per plate of callus.

After the bombardment, calli were transferred back to the fresh callusdevelopment medium and maintained in the dark at room temperature for a1-week period. The callus was then transferred to growth conditions inthe light at 25° C. to initiate embryo differentiation with theappropriate selection agent, e.g. 250 nM Arsenal, mg/l PPT or 50 mg/Lkanamycin. Shoots resistant to the selection agent appeared and oncerotted were transferred to soil.

Samples of the primary transgenic plants (T0) are analyzed by PCR toconfirm the presence of T-DNA. These results are confirmed by Southernhybridization in which DNA is electrophoresed on a 1% agarose gel andtransferred to a positively charged nylon membrane (Roche Diagnostics).The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labelled probe by PCR, and used as recommended by themanufacturer.

Transgenic T0 ryegrass plants were propagated vegetatively by excisingtillers. The transplanted tillers were maintained in the greenhouse for2 months until well established. The shoots were defoliated and allowedto grow for 2 weeks.

The first drought experiment was conducted in a manner similar to thatdescribed in example 3. The seedlings received no water for a period upto 3 weeks at which time the plant and soil were desiccated. At varioustimes after withholding water, a normal watering schedule was resumed.At one week after resuming watering, the lengths of leaf blades, and thefresh and dry weights of the shoots was determined. At an equivalentdegree of drought stress, tolerant plants were able to resume normalgrowth whereas susceptible plants had died or suffered significantinjury resulting in shorter leaves and less dry matter. Proline contentof the leaves and stomatal aperture were also measured at various timesduring the drought stress. Tolerant plants maintained a lower prolinecontent and a greater stomatal aperture than the non-transgenic controlplants.

A second experiment imposing drought stress on the transgenic plants wasby treatment with a solution of PEG as described in the previousexamples. Tolerance of salinity and cold were measured using methods asdescribed in example 3. Plants that had tolerance to salinity or coldhad higher seed yields, photosynthesis and dry matter production thansusceptible plants.

Example 6 Engineering Stress-Tolerant Soybean Plants by Over-ExpressingStress Related Genes from Saccharomyces Cerevisiae or E. Coli orBrassica Napus, Glycine Max, and Oryza Sativa

Soybean was transformed according to the following modification of themethod described in the Texas A&M patent U.S. Pat. No. 5,164,310.Several commercial soybean varieties are amenable to transformation bythis method. The cultivar Jack (available from the Illinois SeedFoundation) is a commonly used for transformation. Seeds were sterilizedby immersion in 70% (v/v) ethanol for 6 min and in 25% commercial bleach(NaOCl) supplemented with 0.1% (v/v) Tween for 20 min, followed byrinsing 4 times with sterile double distilled water. Seven-day seedlingswere propagated by removing the radicle, hypocotyl and one cotyledonfrom each seedling. Then, the epicotyl with one cotyledon wastransferred to fresh germination media in petri dishes and incubated at25° C. under a 16-hr photoperiod (approx. 100 μE-m-2s-1) for threeweeks. Axillary nodes (approx. 4 mm in length) were cut from 3-4week-old plants. Axillary nodes were excised and incubated inAgrobacterium LBA4404 culture.

Many different binary vector systems have been described for planttransformation (e.g. An, G. in Agrobacterium Protocols. Methods inMolecular Biology vol 44, pp 47-62, Gartland K M A and M R Davey eds.Humana Press, Totowa, N.J.). Many are based on the vector pBIN19described by Bevan (Nucleic Acid Research. 1984. 12:8711-8721) thatincludes a plant gene expression cassette flanked by the left and rightborder sequences from the Ti plasmid of Agrobacterium tumefaciens. Aplant gene expression cassette consists of at least two genes—aselection marker gene and a plant promoter regulating the transcriptionof the cDNA or genomic DNA of the trait gene. Various selection markergenes can be used including the Arabidopsis gene encoding a mutatedacetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. Nos. 5,767,3666 and6,225,105). Similarly, various promoters can be used to regulate thetrait gene to provide constitutive, developmental, tissue orenvironmental regulation of gene transcription. In this example, the 34Spromoter (GenBank Accession numbers M59930 and X16673) was used toprovide constitutive expression of the trait gene.

After the co-cultivation treatment, the explants were washed andtransferred to selection media supplemented with 500 mg/L timentin.Shoots were excised and placed on a shoot elongation medium. Shootslonger than 1 cm were placed on rooting medium for two to four weeksprior to transplanting to soil.

The primary transgenic plants (T0) were analyzed by PCR to confirm thepresence of T-DNA. These results were confirmed by Southernhybridization in which DNA is electrophoresed on a 1% agarose gel andtransferred to a positively charged nylon membrane (Roche Diagnostics).The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labelled probe by PCR, and used as recommended by themanufacturer.

Tolerant plants had higher seed yields, maintained their stomatalaperture and showed only slight changes in osmotic potential and prolinelevels, whereas the susceptible non-transgenic control plants closedtheir stomata and exhibited increased osmotic potential and prolinelevels.

Tolerance of drought, salinity and cold were measured using methods asdescribed in example 3. Plants that had tolerance to salinity or coldhad higher seed yields, photosynthesis and dry matter production thansusceptible plants.

Example 7 Engineering Stress-Tolerant Rapeseed/Canola Plants byOver-Expressing Stress Related Genes from Saccharomyces Cerevisiae or E.Coli or Brassica Napus, Glycine Max, and Oryza Sativa

Cotyledonary petioles and hypocotyls of 5-6 day-old young seedlings wereused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can be used.

Agrobacterium tumefaciens LBA4404 containing a binary vector was usedfor canola transformation. Many different binary vector systems havebeen described for plant transformation (e.g. An, G. in AgrobacteriumProtocols. Methods in Molecular Biology vol 44, pp 47-62, Gartland K M Aand M R Davey eds. Humana Press, Totowa, N.J.). Many are based on thevector pBIN19 described by Bevan (Nucleic Acid Research. 1984.12:8711-8721) that includes a plant gene expression cassette flanked bythe left and right border sequences from the Ti plasmid of Agrobacteriumtumefaciens. A plant gene expression cassette consists of at least twogenes—a selection marker gene and a plant promoter regulating thetranscription of the cDNA or genomic DNA of the trait gene. Variousselection marker genes can be used including the Arabidopsis geneencoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat.Nos. 5,767,3666 and 6,225,105). Similarly, various promoters can be usedto regulate the trait gene to provide constitutive, developmental,tissue or environmental regulation of gene transcription. In thisexample, the 34S promoter (GenBank Accession numbers M59930 and X16673)was used to provide constitutive expression of the trait gene.

Canola seeds were surface-sterilized in 70% ethanol for 2 min., and thenin 30% Clorox with a drop of Tween-20 for 10 min, followed by threerinses with sterilized distilled water. Seeds were then germinated invitro 5 days on half strength MS medium without hormones, 1% sucrose,0.7% Phytagar at 23° C., 16 hr. light. The cotyledon petiole explantswith the cotyledon attached were excised from the in vitro seedlings,and inoculated with Agrobacterium by dipping the cut end of the petioleexplant into the bacterial suspension. The explants were then culturedfor 2 days on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7%Phytagar at 23° C., 16 hr light. After two days of co-cultivation withAgrobacterium, the petiole explants were transferred to MSBAP-3 mediumcontaining 3 mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l)for 7 days, and then cultured on MSBAP-3 medium with cefotaxime,carbenicillin, or timentin and selection agent until shoot regeneration.When the shoots were 5-10 mm in length, they were cut and transferred toshoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots ofabout 2 cm in length were transferred to the rooting medium (MSO) forroot induction.

Samples of the primary transgenic plants (T0) were analyzed by PCR toconfirm the presence of T-DNA. These results were confirmed by Southernhybridization in which DNA is electrophoresed on a 1% agarose gel andtransferred to a positively charged nylon membrane (Roche Diagnostics).The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labelled probe by PCR, and used as recommended by themanufacturer.

The transgenic plants were then evaluated for their improved stresstolerance according to the method described in Example 3. Tolerantplants had higher seed yields, maintained their stomatal aperture andshowed only slight changes in osmotic potential and proline levels,whereas the susceptible non-transgenic control plants closed theirstomata and exhibited increased osmotic potential and proline levels.

Tolerance of drought, salinity and cold were measured using methods asdescribed in the previous example 3. Plants that had tolerance tosalinity or cold had higher seed yields, photosynthesis and dry matterproduction than susceptible plants.

Example 8 Engineering Stress-Tolerant Corn Plants by Over-ExpressingStress Related Genes from Saccharomyces Cerevisiae or E. Coli orBrassica Napus, Glycine Max, and Oryza Sativa

Transformation of maize (Zea Mays L.) is performed with a modificationof the method described by Ishida et al. (1996. Nature Biotech14745-50).

Transformation is genotype-dependent in corn and only specific genotypesare amenable to transformation and regeneration. The inbred line A188(University of Minnesota) or hybrids with A188 as a parent are goodsources of donor material for transformation (Fromm et al. 1990 Biotech8:833-839), but other genotypes can be used successfully as well. Earsare harvested from corn plants at approximately 11 days afterpollination (DAP) when the length of immature embryos is about 1 to 1.2mm. Immature embryos are co-cultivated with Agrobacterium tumefaciensthat carry “super binary” vectors and transgenic plants are recoveredthrough organogenesis. The super binary vector system of Japan Tobaccois described in WO patents WO94/00977 and WO95/06722. Vectors wereconstructed as described. Various selection marker genes can be usedincluding the maize gene encoding a mutated acetohydroxy acid synthase(AHAS) enzyme (U.S. Pat. No. 6,025,541). Similarly, various promoterscan be used to regulate the trait gene to provide constitutive,developmental, tissue or environmental regulation of gene transcription.In this example, the 34S promoter (GenBank Accession numbers M59930 andX16673) was used to provide constitutive expression of the trait gene.

Excised embryos are grown on callus induction medium, then maizeregeneration medium, containing imidazolinone as a selection agent. ThePetri plates are incubated in the light at 25° C. for 2-3 weeks, oruntil shoots develop. The green shoots are transferred from each embryoto maize rooting medium and incubated at 25° C. for 2-3 weeks, untilroots develop. The rooted shoots are transplanted to soil in thegreenhouse. T1 seeds are produced from plants that exhibit tolerance tothe imidazolinone herbicides and which are PCR positive for thetransgenes.

The T1 transgenic plants were then evaluated for their improved stresstolerance according to the method described in Example 3. The T1generation of single locus insertions of the T-DNA will segregate forthe transgene in a 3:1 ratio. Those progeny containing one or two copiesof the transgene are tolerant of the imidazolinone herbicide, andexhibit greater tolerance of drought stress than those progeny lackingthe transgenes. Tolerant plants had higher seed yields, maintained theirstomatal aperture and showed only slight changes in osmotic potentialand proline levels, whereas the susceptible non-transgenic controlplants closed their stomata and exhibited increased osmotic potentialand proline levels. Homozygous T2 plants exhibited similar phenotypes.Hybrid plants (F1 progeny) of homozygous transgenic plants andnon-transgenic plants also exhibited increased environmental stresstolerance.

Tolerance of salinity and cold were measured using methods as describedin the previous example 3. Plants that had tolerance to drought,salinity or cold had higher seed yields, photosynthesis and dry matterproduction than susceptible plants.

Example 9 Engineering Stress-Tolerant Wheat Plants by Over-ExpressingStress Related Genes from Saccharomyces Cerevisiae or E. Coli, BrassicaNapus, Glycine Max, or Oryza Sativa

Transformation of wheat is performed with the method described by Ishidaet al. (1996 Nature Biotech. 14745-50). The cultivar Bobwhite (availablefrom CYMMIT, Mexico) is commonly used in transformation. Immatureembryos are co-cultivated with Agrobacterium tumefaciens that carry“super binary” vectors, and transgenic plants are recovered throughorganogenesis. The super binary vector system of Japan Tobacco isdescribed in WO patents WO94/00977 and WO95/06722. Vectors wereconstructed as described. Various selection marker genes can be usedincluding the maize gene encoding a mutated acetohydroxy acid synthase(AHAS) enzyme (U.S. Pat. No. 6,025,541). Similarly, various promoterscan be used to regulate the trait gene to provide constitutive,developmental, tissue or environmental regulation of gene transcription.In this example, the 34S promoter (GenBank Accession numbers M59930 andX16673) was used to provide constitutive expression of the trait gene.

After incubation with Agrobacterium, the embryos are grown on callusinduction medium, then regeneration medium, containing imidazolinone asa selection agent. The Petri plates are incubated in the light at 25° C.for 2-3 weeks, or until shoots develop. The green shoots are transferredfrom each embryo to rooting medium and incubated at 25° C. for 2-3weeks, until roots develop. The rooted shoots are transplanted to soilin the greenhouse. T1 seeds are produced from plants that exhibittolerance to the imidazolinone herbicides and which are PCR positive forthe transgenes.

The T1 transgenic plants were then evaluated for their improved stresstolerance according to the method described in the previous example 3.The T1 generation of single locus insertions of the T-DNA will segregatefor the transgene in a 3:1 ratio. Those progeny containing one or twocopies of the transgene are tolerant of the imidazolinone herbicide, andexhibit greater tolerance of drought stress than those progeny lackingthe transgenes. Tolerant plants had higher seed yields, maintained theirstomatal aperture and showed only slight changes in osmotic potentialand proline levels, whereas the susceptible non-transgenic controlplants closed their stomata and exhibited increased osmotic potentialand proline levels. Homozygous T2 plants exhibited similar phenotypes.Tolerance of salinity and cold were measured using methods as describedin the previous examples. Plants that had tolerance to drought, salinityor cold had higher seed yields, photosynthesis and dry matter productionthan susceptible plants.

Example 14 Identification of Identical and Heterologous Genes

Gene sequences can be used to identify identical or heterologous genesfrom cDNA or genomic libraries. Identical genes (e.g. full-length cDNAclones) can be isolated via nucleic acid hybridization using for examplecDNA libraries. Depending on the abundance of the gene of interest,100,000 up to 1,000,000 recombinant bacteriophages are plated andtransferred to nylon membranes. After denaturation with alkali, DNA isimmobilized on the membrane by e.g. UV cross linking. Hybridization iscarried out at high stringency conditions. In aqueous solution,hybridization and washing is performed at an ionic strength of 1 M NaCland a temperature of 68° C.

Hybridization probes are generated by e.g. radioactive (³²P) nicktranscription labeling (High Prime, Roche, Mannheim, Germany). Signalsare detected by autoradiography.

Partially identical or heterologous genes that are related but notidentical can be identified in a manner analogous to the above-describedprocedure using low stringency hybridization and washing conditions. Foraqueous hybridization, the ionic strength is normally kept at 1 M NaClwhile the temperature is progressively lowered from 68 to 42° C.

Isolation of gene sequences with homology (or sequenceidentity/similarity) only in a distinct domain of (for example 10-20amino acids) can be carried out by using synthetic radio labeledoligonucleotide probes. Radiolabeled oligonucleotides are prepared byphosphorylation of the 5-prime end of two complementary oligonucleotideswith T4 polynucleotide kinase. The complementary oligonucleotides areannealed and ligated to form concatemers. The double strandedconcatemers are than radiolabeled by, for example, nick transcription.Hybridization is normally performed at low stringency conditions usinghigh oligonucleotide concentrations.

Oligonucleotide Hybridization Solution: 6×SSC

0.01 M sodium phosphate

1 mM EDTA (pH 8) 0.5% SDS

100 μg/ml denatured salmon sperm DNA0.1% nonfat dried milk

During hybridization, temperature is lowered stepwise to 5-10° C. belowthe estimated oligonucleotide T_(m) or down to room temperature followedby washing steps and autoradiography. Washing is performed with lowstringency such as 3 washing steps using 4×SSC. Further details aredescribed by Sambrook, J. et al., 1989, “Molecular Cloning: A LaboratoryManual,” Cold Spring Harbor Laboratory Press or Ausubel, F. M. et al.,1994, “Current Protocols in Molecular Biology,” John Wiley & Sons.

Example 15 Identification of Identical Genes by Screening ExpressionLibraries With Antibodies

c-DNA clones can be used to produce recombinant polypeptide for examplein E. coli (e.g. Qiagen QIAexpress pQE system). Recombinant polypeptidesare then normally affinity purified via Ni-NTA affinity chromatography(Qiagen). Recombinant polypeptides are then used to produce specificantibodies for example by using standard techniques for rabbitimmunization. Antibodies are affinity purified using a Ni-NTA columnsaturated with the recombinant antigen as described by Gu et al., 1994,BioTechniques 17:257-262. The antibody can than be used to screenexpression cDNA libraries to identify identical or heterologous genesvia an immunological screening (Sambrook, J. et al., 1989, “MolecularCloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press orAusubel, F. M. et al., 1994, “Current Protocols in Molecular Biology”,John Wiley & Sons).

Example 16 In Vivo Mutagenesis

In vivo mutagenesis of microorganisms can be performed by passage ofplasmid (or other vector) DNA through E. coli or other microorganisms(e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) whichare impaired in their capabilities to maintain the integrity of theirgenetic information. Typical mutator strains have mutations in the genesfor the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; forreference, see Rupp, W. D., 1996, DNA repair mechanisms, in: Escherichiacoli and Salmonella, p. 2277-2294, ASM: Washington.) Such strains arewell known to those skilled in the art. The use of such strains isillustrated, for example, in Greener, A. and Callahan, M., 1994,Strategies 7: 32-34. Transfer of mutated DNA molecules into plants ispreferably done after selection and testing in microorganisms.Transgenic plants are generated according to various examples within theexemplification of this document.

Example 17 Identification and Analysis of YNLO90W Homologs

Transgenic Arabidopsis plants overexpressing YNL 090W (ORF 3165) lived4.33 days longer than the wild type control under drought conditions.The protein sequence of YNLO90W was used to identify related genesequences of expressed sequence tags (ESTs) proprietary librariesconstructed from Oryza sativa cv. Noppon-Brarre (a japonica rice),Brassica napus cv. “AC Excel” “Quantum” and “Cresor” (canola), andGlycine max cv. Resuick (soybean) by Blast analysis (Altschul S F, GishW, Miller W, Myers E W, Lipman D J. 1990 J Mol Biol 21593:403-10).

The plant cDNA sequences were translated into a predicated amino acidsequences, and relationship among the amino acids sequences wasdetermined by sequence alignment using the clustal W alogorithm inVector NTI Version 7 as shown in the drawing.

They have 75% similarity overall. In general, proteins having conserveddomains referred to as I (GXXXGKT), II (DXXG), III (VGTK), IV (EXSS),and E (FXXXYXX), are classified as small GTPases. All five domains inthe proteins as shown in the drawing are more conserved than in otherknown small GTPases, particularly Domain I (KXVXXGDXXXGKT) (SEQ ID NO:557), Domain E (FXXXYXXTV) (SEQ ID NO: 558), Domain II (WDTAGQE) (SEQ IDNO: 559) and Domain III (VGTKXDL) (SEQ ID NO: 560). In addition,polybasic amino acids in C terminus are shown in the drawing, as well asa C-terminal CAAL domain (wherein A is an aliphatic amino acid), whichis the signature sequence for post translational modification by theenzyme geranylgeranyltransferase I. Another important Rho-specificfeature was shown in most proteins in the drawing is the highlyconserved amino acids between Domain II and Domain III.

All the references described above are incorporated by reference in itsentirety for all useful purposes.

While there is shown and described certain specific examples embodyingthe invention, it will be manifest to those skilled in the art thatvarious modifications and rearrangements of the parts may be madewithout departing from the spirit and scope of the underlying inventiveconcept and that the same is not limited to the particular examplesherein shown and described.

1. A nucleic acid construct comprising a Stress-Related Protein (SRP)coding nucleic acid operably linked to at least one promoter whichconfers expression of said SRP coding nucleic acid in a plant or plantcell, and optionally one or more additional regulatory elements, whereinsaid SRP coding nucleic acid is selected from the group consisting of:(a) a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275,277, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305,307, 309, 311, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416,418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444,446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472,474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500,502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 525, 527,529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, or 555;(b) a nucleic acid molecule encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 313, 315, 317, 319, 321, 323, 325,327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353,355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381,383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409,411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465,467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493,495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521,523, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550,552, 554, or 556; and (c) a nucleic acid molecule encoding a polypeptidecomprising an amino acid sequence having at least 90% sequence identitywith the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, 266, 268, 270, 272, 274, 276, 278, 282, 284, 286, 288, 290,292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 313, 315, 317, 319,321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403,405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431,433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459,461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487,489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515,517, 519, 521, 523, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544,546, 548, 550, 552, 554, or 556, and conferring increased toleranceand/or resistance to the environmental stress in the transgenic plant orplant cell as compared to a corresponding non-transformed wild typeplant or plant cell, wherein expression of said SRP coding nucleic acidin a plant or plant cell confers increased tolerance and/or resistanceto an environmental stress as compared to a corresponding wild typeplant or plant cell, and wherein the environmental stress comprisesdrought stress.
 2. A transgenic plant or plant cell with increasedtolerance and/or resistance to an environmental stress as compared to acorresponding non-transformed wild type plant or plant cell, wherein thetransgenic plant or plant cell is transformed with the nucleic acidconstruct of claim 1, wherein expression of said SRP coding nucleic acidconfers increased tolerance and/or resistance to the environmentalstress in the transgenic plant or plant cell as compared to thecorresponding non-transformed wild type plant or plant cell, and whereinthe environmental stress comprises drought stress.
 3. The transgenicplant or plant cell of claim 2, wherein the transgenic plant is amonocotyledonous plant, a dicotyledonous plant, or a gymnosperm plant,or wherein the transgenic plant cell is obtained from a monocotyledonousplant, a dicotyledonous plant, or a gymnosperm plant.
 4. A transgenicplant generated from the transgenic plant cell of claim
 2. 5. A seedproduced by the transgenic plant of claim 2, wherein the transgenicplant is a monocotyledonous plant, a dicotyledonous plant, or agymnosperm plant, and wherein the transgenic plant is true breeding forincreased tolerance and/or resistance to the environmental stress.
 6. Aprogeny or a plant part of the transgenic plant of claim 2, wherein saidprogeny or said plant part comprises the SRP coding nucleic acid.
 7. Thetransgenic plant or plant cell of claim 2, wherein the SRP codingnucleic acid is selected from the group consisting of: (a) a nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO: 255; (b)a nucleic acid molecule encoding a polypeptide comprising the amino acidsequence of SEQ ID NO: 256; and (c) a nucleic acid molecule encoding apolypeptide comprising an amino acid sequence having at least 90%sequence identity with the amino acid sequence of SEQ ID NO: 256 andconferring increased tolerance and/or resistance to the environmentalstress in the transgenic plant cell as compared to a correspondingnon-transformed wild type plant cell.
 8. A method of producing atransgenic plant with increased tolerance and/or resistance to anenvironmental stress as compared to a corresponding non-transformed wildtype plant, comprising: (i) transforming a plant cell with the nucleicacid construct of claim 1 or a vector comprising said nucleic acidconstruct, (ii) generating from the plant cell a transgenic plant withincreased tolerance and/or resistance to an environmental stress ascompared to a corresponding non-transformed wild type plant, whereinexpression of said SRP coding nucleic acid confers increased toleranceand/or resistance to the environmental stress in the transgenic plant ascompared to a corresponding non-transformed wild type plant, and whereinthe environmental stress comprises drought stress.
 9. The method ofclaim 8, wherein the plant cell is obtained from a monocotyledonousplant, a dicotyledonous plant, or a gymnosperm plant.
 10. The method ofclaim 8, wherein the SRP coding nucleic acid is selected from the groupconsisting of: (a) a nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO: 255; (b) a nucleic acid molecule encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 256; and(c) a nucleic acid molecule encoding a polypeptide comprising an aminoacid sequence having at least 90% sequence identity with the amino acidsequence of SEQ ID NO: 256 and conferring increased tolerance and/orresistance to the environmental stress in the transgenic plant cell ascompared to a corresponding non-transformed wild type plant cell.
 11. Amethod of increasing tolerance and/or resistance to an environmentalstress in a plant, comprising increasing the level of expression of anSRP in a plant by introducing the nucleic acid construct of claim 1 intosaid plant, wherein expression of the SRP coding nucleic acid confersincreased tolerance and/or resistance to the environmental stress in theplant as compared to a corresponding non-transformed wild type plant,and wherein the environmental stress comprises drought stress.
 12. Themethod of claim 11, wherein the plant is a monocotyledonous plant, adicotyledonous plant, or a gymnosperm plant.
 13. The method of claim 11,wherein the SRP coding nucleic acid is selected from the groupconsisting of: (a) a nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO: 255; (b) a nucleic acid molecule encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 256; and(c) a nucleic acid molecule encoding a polypeptide comprising an aminoacid sequence having at least 90% sequence identity with the amino acidsequence of SEQ ID NO: 256 and conferring increased tolerance and/orresistance to the environmental stress in the transgenic plant cell ascompared to a corresponding non-transformed wild type plant cell.
 14. Aplant or plant cell comprising a nucleic acid construct conferringexpression of an SRP coding nucleic acid in said plant or plant cell,wherein the nucleic acid construct comprises the SRP coding nucleic acidand one or more regulatory elements, wherein expression of the SRPcoding nucleic acid in the plant or plant cell results in increasedtolerance and/or resistance to an environmental stress as compared to acorresponding non-transformed wild type plant or plant cell, wherein theSRP coding nucleic acid is selected from the group consisting of: (a) anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275,277, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305,307, 309, 311, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416,418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444,446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472,474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500,502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 525, 527,529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, or 555;(b) a nucleic acid molecule encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 313, 315, 317, 319, 321, 323, 325,327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353,355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381,383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409,411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465,467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493,495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521,523, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550,552, 554, or 556; and (c) a nucleic acid molecule encoding a polypeptidecomprising an amino acid sequence having at least 90% sequence identitywith the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, 266, 268, 270, 272, 274, 276, 278, 282, 284, 286, 288, 290,292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 313, 315, 317, 319,321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403,405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431,433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459,461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487,489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515,517, 519, 521, 523, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544,546, 548, 550, 552, 554, or 556, and conferring increased toleranceand/or resistance to the environmental stress in the plant or plant cellas compared to a corresponding non-transformed wild type plant or plantcell, and wherein the environmental stress comprises drought stress. 15.The plant or plant cell of claim 14, wherein the plant is amonocotyledonous plant, a dicotyledonous plant, or a gymnosperm plant,or wherein the transgenic plant cell is obtained from a monocotyledonousplant, a dicotyledonous plant, or a gymnosperm plant.
 16. A seedproduced by the plant of claim 14, or a progeny or a plant part of saidplant, wherein said seed, said progeny, or said plant part comprises theSRP coding nucleic acid.
 17. The plant or plant cell of claim 14,wherein the SRP coding nucleic acid is selected from the groupconsisting of: (a) a nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO: 255; (b) a nucleic acid molecule encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 256; and(c) a nucleic acid molecule encoding a polypeptide comprising an aminoacid sequence having at least 90% sequence identity with the amino acidsequence of SEQ ID NO: 256 and conferring increased tolerance and/orresistance to the environmental stress in the transgenic plant cell ascompared to a corresponding non-transformed wild type plant cell.
 18. Amethod of producing a transgenic plant with increased tolerance and/orresistance to an environmental stress as compared to a correspondingnon-transformed wild type plant, comprising: (i) transforming a plant ora plant cell with a SRP coding nucleic acid or a vector comprising saidSRP coding nucleic acid; (ii) optionally generating from the plant cella transgenic plant; and (iii) selecting a transgenic plant withincreased tolerance and/or resistance to an environmental stress ascompared to a corresponding non-transformed wild type plant, wherein theSRP coding nucleic acid is selected from the group consisting of: (a) anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247,249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275,277, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305,307, 309, 311, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360,362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416,418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444,446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472,474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500,502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 525, 527,529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, or 555;(b) a nucleic acid molecule encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,268, 270, 272, 274, 276, 278, 282, 284, 286, 288, 290, 292, 294, 296,298, 300, 302, 304, 306, 308, 310, 313, 315, 317, 319, 321, 323, 325,327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353,355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381,383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409,411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465,467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493,495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521,523, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550,552, 554, or 556; and (c) a nucleic acid molecule encoding a polypeptidecomprising an amino acid sequence having at least 90% sequence identitywith the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232,234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,262, 264, 266, 268, 270, 272, 274, 276, 278, 282, 284, 286, 288, 290,292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 313, 315, 317, 319,321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403,405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431,433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459,461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487,489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515,517, 519, 521, 523, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544,546, 548, 550, 552, 554, or 556, and conferring increased toleranceand/or resistance to drought stress in the transgenic plant as comparedto a corresponding non-transformed wild type plant, and wherein theenvironmental stress comprises drought stress.
 19. The method of claim18, wherein the plant is a monocotyledonous plant, a dicotyledonousplant, or a gymnosperm plant, or wherein the plant cell is obtained froma monocotyledonous plant, a dicotyledonous plant, or a gymnosperm plant.20. The method of claim 18, wherein the SRP coding nucleic acid isselected from the group consisting of: (a) a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO: 255; (b) a nucleic acidmolecule encoding a polypeptide comprising the amino acid sequence ofSEQ ID NO: 256; and (c) a nucleic acid molecule encoding a polypeptidecomprising an amino acid sequence having at least 90% sequence identitywith the amino acid sequence of SEQ ID NO: 256 and conferring increasedtolerance and/or resistance to the environmental stress in thetransgenic plant cell as compared to a corresponding non-transformedwild type plant cell.